Treating hyperinsulinemia by killing beta cells with cytotoxic gene

ABSTRACT

This invention relates to a recombinant nucleic acid for an RIP-tk (rat insulin promoter-thymidine kinase) construct that selectively targets insulin secreting cells, such as β-cells, PDX-1 positive human pancreatic ductal carcinomas, and other cells containing certain transcription factors. The present invention is useful in the treatment of hyperinsulinemia by targeting β-cells that overproduce insulin with a cytotoxic gene.

PRIOR RELATED APPLICATIONS

This application claims the benefit of U.S. application Ser. No.10/656,450 filed on Sep. 5, 2003 which is a continuation of U.S.application Ser. No. 09/686,631 filed on Oct. 11, 2000. It also claimsthe benefit of U.S. Provisional Patent Application No. 60/161,109, filedOct. 22, 1999, and U.S. Provisional Patent Application No. 60/224,382,filed Aug. 9, 2000.

FEDERALLY SPONSORED RESEARCH STATEMENT

This invention was made in part with United States Government supportunder grant number 2 RO1 DK 46441-09 awarded by the National Instituteof Health, and the United States Government has certain rights in theinvention.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

This invention relates to selective targeting of cells with cytotoxicgenes using fingerprinting promoter driven specific cytotoxic geneticconstructs and transcription factors, and to methods for using theseconstructs and transcription factors to treat diseases of the pancreas,and, in particular, to an RIP-tk (rat insulin promoter-thymidine kinase)construct that selectively targets insulin secreting cells, such as beta(β) cells, and certain human pancreatic ductal carcinoma cells, to causecell death.

BACKGROUND OF THE INVENTION

β-cell adenomas (insulinomas) are the most common of the islet celltumors. Ninety percent of β-cell tumors are benign, however morbidityassociated with their removal is significant. Malignant insulinomas havea 63% five-year recurrence rate with an average survival less than fouryears (Proye, C, 68 AUST. N.Z.J. SURG. 90-100 (1998)). Furthermore,there is no effective medical treatment for the devastating symptomsassociated with hyperinsulinemia as a result of either insulinoma ornesidioblastosis (idiopathic hyperinsulinemia). Pancreatic ductaladenocarcinoma (PDA) likewise remains a devastating disease with a lessthan three percent five year survival rate.

Over 28,000 patients will be diagnosed with pancreatic cancer this yearof which over 27,000 will die of their disease within five years (Yeo Cj, et al., Neoplasms of the Pancreas Exocrine Tumors in SABISTONTEXTBOOK OF SURGERY 1171-1175 (Sabiston D C, et al. eds., 1997)). Themajority of pancreatic tumors arise from ductular cells, resemblingcells found in the early embryonic pancreas. Currently, only surgeryoffers any chance for a cure and the priority of the time the cancer hasspread before it is detected.

Cancer specific promoters are being identified and are being used in aneffort to modify the expression of thymidine kinase in tumor cells(Tanaka T, et al., 231 BIOCHEM. BIOPHYS. RES. COMMUN. 775-779 (1997);DiMaio J, et al., 116[2] SURG. 205-213 (1994); Osaki T, et al., 54CANCER RES. 5258-5261 (1994); Kaneko S, et al., 55 CANCER RES. 5283-5287(1995); Robertson M, et al., 5[5] CANCER GENE THER. 331-336 (1998);Siders W, et al., 5[5] CANCER GENE THER. 181-291(1998); Vandier D., etal., 58 CANCER RES. 4577-4590 (1998)). However, these therapies havelimitations due to either the weakness of the promoter or the tissuespecificity of its activation. The herpes simplex thymidine kinase (HSVtk) gene, under the transcriptional control of a ubiquitous promoter,has been introduced into a host and caused significant cell death in thepresence of ganciclovir (Bonnekoh B. et al., 104 J. INVESTIG. DERMAT.313-317 (1995); Al-Hendy A, et al., 43 GYNEC. OBST. INVESTIG. 268-275(1997); Eastham J, et al., 7 HUMAN GENE THER. 515-523 (1996); Chen S-H,et al., 91 PROC. NATL. ACAD. SCI. USA 3054-3057 (1994); Tong X, et al.,61 GYNEC. ONCOL. 175-179 (1996). Ganciclovir (GCV), an analogue ofguanosine, requires both mammalian and viral tk to become active. Inviral thymidine kinase containing cells, GCV is phosphorylated into anintermediate that kills dividing cells by inhibiting DNA synthesis andacting as a chain terminator (Mathews T, et al., 10 REV. INFECT. DIS.180-192 (1992); Moolten Fla., 50 CANCER RES. 7820-7825 (1986)). However,thymidine kinase with a ubiquitous promoter is not cell specific,limiting its use as a cytotoxic agent.

With the identification of tissue specific promoters, one can targettherapies and selectively turn on genes in specific cell types,important goals in gene therapy. Cell specific strategies depend on acell specific promoter that can activate the suicide gene only in thetargeted tumor. To accomplish this, activation of the promoter-suicideconstruct should require the presence of transcription factors in thetargeted tumor that will activate the promoter. In addition, aneffective gene delivery system is needed. However, prior to the presentinvention, a method to express selected genes solely in P cells andother cells of pancreatic origin had not been developed. Such a methodwould provide a useful tool for development of treatment for insulinoma,nesidioblastosis and other pancreatic cancers.

Currently, there is no effective treatment for pancreatic β-cell tumorsor pancreatic ductal adenocarcinomas. Consequently, there is a need foran effective and selective treatment for these diseases, as well asother diseases due to abnormal pancreatic cells.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides a novelrecombinant nucleic acid comprising an RIP-tk construct useful for theselective targeting and ablation of cells, such as cells comprising oneor more specific transcription factors. The invention is particularlyuseful for the treatment of pancreatic cancer and other diseases of oraffecting the pancreas.

Accordingly, one embodiment of the invention is directed to a method forselectively expressing a target gene in a pancreatic cell comprisingdelivering to the cell an effective amount of an agent containing arecombinant nucleic acid sequence, the sequence comprising an insulinpromoter, such as a rat insulin promoter, operatively linked to thetarget gene. The cell may naturally contain or be cotransfected with oneor more insulin promoter transcription factors selected from the groupconsisting of BETA-2, GATA4, E47 and PDX-1.

Another embodiment of the invention is directed to a method forselectively ablating pancreatic cells in an individual comprisingadministering to the individual an effective amount of an agentcomprising a recombinant nucleic acid sequence. The recombinant nucleicacid sequence preferably comprises a cytotoxic gene operatively linkedto an insulin promoter, such as the rat insulin promoter. The pancreaticcells preferably express one or more transcription factors selected fromthe group consisting of BETA-2, GATA4, E47 and PDX-1. Preferably, thecytotoxic gene is a nucleic acid encoding viral thymidine kinase (tk),and the method further comprises the step of administering ganciclovir,acyclovir, FIAU or 6-methoxypurine arabinoside to the individual in anamount effective to ablate the cells.

Another embodiment is directed to a method for selectively ablating atarget in an individual comprising transfecting the individual with anagent comprising a recombinant nucleic acid sequence comprising athymidine kinase gene operatively linked to an insulin promoter, such asa rat insulin promoter, and administering to the individual an effectiveamount of ganciclovir, acyclovir, FIAU or 6-methoxypurine arabinoside inan amount sufficient to cause ablation of the target. Preferably, thetarget comprises cells that naturally express or are cotransfected withone or more rat insulin promoter transcription factors selected from thegroup consisting of BETA-2, GATA4, E47 and PDX-1.

Another embodiment is directed to a method for the production of aprotein in a cell comprising delivering a nucleic acid molecule to thecell, wherein the nucleic acid molecule comprises an insulin promoteroperatively linked to a structural nucleic acid sequence encoding theprotein, and the cell comprises the PDX-1 transcription factor afterdelivery of the nucleic acid molecule.

Another embodiment of the invention is directed to a method for ablatingcells in an individual comprising delivering an agent to the individual,wherein the agent comprises a nucleic acid molecule, and the nucleicacid molecule comprises an insulin promoter operatively linked to astructural nucleic acid sequence encoding a cytotoxic protein.

Another embodiment of the invention is directed to a method for treatinga metabolic disease, such as hypoglycemia or hyperinsulinemia, in anindividual comprising administering to the individual an effectiveamount of an agent comprising a recombinant nucleic acid sequencecomprising a cytotoxic gene operatively linked to an insulin promoter.Preferably, the cytotoxic gene encodes thymidine kinase and the methodfurther comprises the step of administering to the individual an agent,such as ganciclovir.

Another embodiment of the invention is directed to a method of treatinga metabolic disease in an individual comprising delivering an agent tothe individual, wherein the agent comprises a nucleic acid molecule, andthe nucleic acid molecule comprises an insulin promoter operativelylinked to a structural nucleic acid sequence encoding a cytotoxicprotein.

Another embodiment is directed to a composition for selectively causingregression or ablation of a pancreatic cell comprising a recombinantnucleic acid sequence which comprises a thymidine kinase geneoperatively linked to an insulin promoter, such as the rat insulinpromoter.

Another embodiment of the invention is directed to an isolated nucleicacid molecule comprising an insulin promoter operatively linked to astructural nucleic acid sequence encoding a cytotoxic protein.

Another embodiment of the invention is directed to a kit comprising anisolated nucleic acid molecule, wherein the isolated nucleic acidmolecule comprises an insulin promoter operatively linked to astructural nucleic acid sequence encoding a cytotoxic protein.Preferably, the nucleic acid molecule is contained in a first container,and the kit further comprises one or more agents selected from the groupconsisting of ganciclovir, acyclovir, FIAU, and 6-methoxypurinearabinoside contained in the same or a second container.

Another embodiment of the invention is directed to a method forincreasing the secretion of insulin in an individual comprising reducingthe concentration of the somatostatin receptor.

Other embodiments and advantages of the invention are set forth in partin the description which follows, and in part, will be obvious from thisdescription, or may be learned from the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a dose response curve for NIT-1 cells given GCV.

FIG. 1B is a dose response curve for F9 cells given GCV.

FIG. 2 is a bar graph showing the percent cell survival of NIT-1 cellstransfected with nothing (C), a hollow vector (V), and RIP-tk withincreasing levels of GCV.

FIG. 3 is a bar graph showing the percent cell survival of F9 cellstransfected with nothing (C), a hollow vector (V), RIP-tk, and MC-1-tkwith increasing levels of GCV.

FIG. 4 is a graph showing blood sugars of ICR/scid mice injected EP with5×10⁶ NIT-1 cells, showing that NIT-I cell tumors were successfullytargeted with the RIP-LacZ gene.

FIG. 5A is a dose response curve for PANC-1 cells given GCV.

FIG. 5B is a dose response curve for CAPAN-1 cells given GCV.

FIG. 5C is a gel electrophoresis for RT-PCR products of PANC-1, CAPAN-1and MIA-1 RNA, with primers specific for PDX-1 and BETA-2.

FIG. 6A is an EMSA of PANC-1 nuclear extract mixed with α³²P dGTPlabeled RIP primer containing a PDX-1 binding site (CTTAAT).

FIG. 6B is an EMSA of CAPAN-1 nuclear extract mixed with α²¹P dGTPlabeled RIP primer containing a PDX-1 binding site (CTAAT).

FIG. 7 is a glucose stimulated insulin versus time curve for 3-month-oldand 12-month-old KO and wt mice.

FIG. 8 is a photograph of stained cells demonstrating the expression ofRIP-LacZ in vivo in PANC-1 cells.

FIG. 9 contains two bar graphs depicting the cell survival percentagesof NIT-1 (left) and F9 (right) cell lines following transfection and GCVtreatment.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following sequence listings form part of the present specificationand are included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these sequences in combination with the description of theinvention presented herein. SEQ ID NO:1 is a fragment of the rat insulinpromoter. SEQ ID NO:2 is the BETA-2 site CANNTG. SEQ ID NO:3 is thePDX-1 site TAAT. SEQ ID NO:4 is the PDX-1 binding site CTTAAT. SEQ IDNO:5 is the mouse PDX-1 primer forward bps 281-300. SEQ ID NO:6 is themouse PDX-1 primer reverse bps 1227-1201. SEQ ID NO:7 is primer forwardbps 944-964 specific for both human and mouse BETA-2 RNA. SEQ ID NO:8 isprimer reverse bps 1227-1207 specific for both human and mouse BETA-2RNA. SEQ ID NO:9 is a probe containing a BETA-2 site and a PDX-1 site.SEQ ID NO:10 is primer forward bps 192-210 for human PDX-1 RNA. SEQ IDNO:11 is primer reverse bps 644-624 for human PDX-1 RNA. SEQ ID NO:12 isPDX-1 binding site CTCCCC. SEQ ID NO:13 is PDX-1 binding site ATATAC.SEQ ID NO:14 is a primer adding a HindIII restriction site. SEQ ID NO:15is a primer adding a Bg1II restriction site.

As embodied and broadly described herein, the present invention isdirected to selective targeting of pancreatic cells with cytotoxic genesusing promoter driven specific cytotoxic genetic constructs andtranscription factors, and to methods for using these constructs andtranscription factors to treat cancer and other diseases.

Specifically, the present invention is directed to an RIP-tk (ratinsulin promoter-thymidine kinase) construct that selectively targetsinsulin secreting cells, such as beta β cells and certain humanpancreatic ductal carcinoma cells (PDX-1 positive), to cause cell death.

Currently, there is no effective medical treatment for pancreaticβ-tumors or insulinomas. Existing therapies which employpromoter-thymidine kinase constructs are not tumor-specific, whichlimits their efficacy. It has been discovered that by using an insulinpromoter coupled to a suicide gene, the efficacy of pancreatic β-celltumor gene therapy may be greatly increased. The present invention isuseful for treatment of insulin secreting tumors as well as in thedevelopment of new adjuvant gene therapies for patients with pancreaticβ-cell tumors or insulinomas. The invention may also be used to treatPDX-1 positive pancreatic ductal cancers and hypoglycemia.

The present invention demonstrates that pancreatic tumor specificcytotoxicity can be achieved through the use of promoter specific genetherapy. For example, it has been discovered that the rat insulinpromoter coupled to the thymidine kinase gene can be used with GCV tospecifically cause cell cytotoxicity in a mouse insulinoma (NIT-1) cellline and in PDX-1 positive human pancreatic ductal cancer cells. Bycoupling rat insulin promoter to a suicide gene, mouse insulinoma cellline (NIT-1 cells) were ablated both in vitro and in vivo with GCV.Similar results were achieved with PDX-1 positive human pancreaticductal cancer cells. As such, it has been discovered that by operativelylinking a suicide gene, such as thymidine kinase, to an insulinpromoter, the thymidine kinase gene will be selectively activated inβ-cell tumors and certain pancreatic ductal cancers, thereby causingtumor cell death.

Furthermore, the cytotoxic effect of RIP-tk may be enhanced in thepresence of RIP transcription factors such as, for example, PDX-1,BETA-2, GATA4, and E47. These transcription factors increase theefficiency of RIP, and are useful in studies of how the promoter works.

As indicated in greater detail in the examples below, it has been orwill be shown that: a) rat insulin promoter (RIP) will drive theexpression of the reporter gene β-galactosidase (LacZ), in vitro, in amouse insulinoma cell line (NIT-1) and in human pancreatic ductaladenocarcinoma cell lines (PANC-1, CAPAN-1); b) NIT-1, PANC-1 andCAPAN-1 specific ablation can be achieved in vitro using thymidinekinase coupled to RIP (RIP-tk) followed by ganciclovir (GCV) treatment;c) RIP transcription factor BETA-2 regulates the expression of RIP-tk inNIT-1 cells while PDX-1 regulates expression in human pancreatic ductalcarcinoma (CAPAN-1 and PANG-1) cell lines; d) the cytotoxic effect ofRIP-tk in vitro can be enhanced by cotransfection of RIP transcriptionfactors, BETA-2, GATA4, PDX-1 and E47; e) RIP will drive the expressionof LacZ in a mouse insulinoma model and in a PDX-1 positive humanpancreatic ductal carcinoma mouse model in vivo; f) insulinoma-specificablation can be achieved in vivo using the RIP-tk gene followed by GCVtreatment; and g) RIP-tk followed by GCV treatment can be used to ablatePDX-1 positive human pancreatic ductal carcinoma in vivo.

β-cell specific cytotoxicity using a rat insulin promoter-thymidinekinase construct: It has been discovered that a RIP-tk construct may beused to selectively target β-cells. The rat insulin promoter (RIP) is astrong cell specific promoter that is activated in cells that produceinsulin. It has been discovered that by incorporating RIP into aconstruct with the suicide gene, thymidine kinase gene (tk), both invitro and in vivo β-cell specific cytotoxicity may be achieved, and thatsuch constructs may be used to selectively kill proliferating β-cells,preventing hypoglycemia and animal death.

The rat insulin promoter (RIP; see Crowe D et al., 9 MOLEC. CELL BIOL.1784-1789 (1989); Ray M, et al., 25 INT. J. PANCREATOL. 157-163 (1999))was studied to determine its usefulness to selectively target NIT-1cells (mouse beta (β) cell tumor line derived from the non-obesediabetic mouse (Hamaguchi D, et al., 40 DIABETES 842-844 (1991)) and todetermine the transcription factors responsible for NIT-1 cell specificactivation of RIP. In addition, the effect of the RIP-tk construct onboth blood glucose levels and life expediency in mice inoculated withNIT-1 cells was evaluated.

As shown in Examples 1, 5 and 6, below, the data confirm that the RIP isa β-cell specific promoter. It has been discovered that β-cell specificcytotoxicity can be accomplished and β cells can be selectively targetedfor destruction using RIP coupled to the suicide gene, thymidine kinase.Further, animals can be rescued from the devastating effects ofhypoglycemia induced by β-cell adenomas (NIT-1 cells) using suchconstructs.

It has been determined that 0.502 kb of RIP (SEQ ID NO: 1) containselements to maximally drive the expression of a gene. These elementsinclude six BETA-2 sites (CANNTG; SEQ ID NO: 2) and three PDX-1 sites(TAAT; SEQ ID NO: 3), one of which contains the sequence CTTAAT (SEQ IDNO: 4), which is the most favorable PDX-1 binding sequence. This smallerfragment allows for the development of smaller constructs. Suchfragments make the creation of the constructs technically easier,increase the transfection efficiency in vitro, and aid in thedevelopment of in vivo gene delivery systems.

In an initial study, REP was used to drive expression of the LacZ genein a variety of cell lines, including NIT-1, F9, 3T3, H411 and CV-1cells. NIT-1 cells form β-cell tumors when injected into the peritoneumof mice with the mice dying of hypoglycemia within one hundred days.Mouse embryonic carcinoma cell line F9, mouse fibroblast cell line 3T3and mouse lung cell line H411 were chosen as control cells because theyrepresent a variety of rodent cell types. Monkey renal CV-1 cells werealso included as a control to represent a higher mammalian species.

Despite the variety of cell types, as shown in Examples 1 and 5, theRIP-LacZ gene failed to display any LacZ gene expression in any of thecontrol cells, despite staining using the RSV-LacZ gene in the samecells. However, in NIT-1 cells, the RIP-LacZ gene consistently expressedbeta-galactosidase protein, confirming that RIP is a β-cell specificpromoter. In addition, significant decrease in cell survival wasobserved in NIT-1 cells transfected with RIP-tk, in vitro (p<0.05,n=48).

RT-PCR was performed looking for the transcription factors BETA-2 andPDX-1 because they have been shown to be responsible for activation ofthe rat insulin promoter in the islets of a rat (Sander M, et al., 75MOL MED. 327-340 (1997)). Messenger RNA for both BETA-2 and PDX-1 wasfound in NIT-1 cells.

Because the mere presence of mRNA for either BETA-2 or PDX-1 in thecells under study does not prove that they are responsible for RIPactivation, EMSA were performed with an oligonucleotide that containedboth a BETA-2 site and a PDX-1 site. A supershift was observed for bothBETA-2 and PDX-1. Specifically, supershift analysis with both BETA-2 andPDX-1 antibodies substantiated that these two transcription factors arein part responsible for RIP activation in NIT-1 cells.

In vivo studies were also performed. In one study, NIT-1 cell tumorswere successfully targeted with the RIP-LacZ gene. In addition, miceinoculated with NIT-1 cells developed clinically relevant tumors withhypoglycemia and death at sixty days post inoculation. However, the invivo delivery of the RIP-tk gene in combination with GCV inhibitedhypoglycemia and animal death (n=30, p<0.05).

In sum, the data demonstrate that a β-cell tumor line can be targeted,in vitro and in vivo, for genetic manipulation using the rat insulinpromoter. As shown in the examples, only the beta (NIT-1) cells stainedblue after X-gal staining (p<0.05, n=16) or had detectable levels ofbeta-galactosidase protein (p<0.05, n=6) in vitro. The RIP-tk geneticconstruct resulted in NIT-1 specific cytotoxicity. F9 cells demonstratedno decrease in cell survival with the RIP-tk gene suggesting that therewas no transcription of the thymidine kinase gene with the rat insulinpromoter in F9 cells. RIP-tk in combination with GCV inhibitedhypoglycemia and death in mice with NIT-1 tumors. Thus, the rat insulinpromoter may be used to achieve β-cell specific cytotoxicity with thetissue specific activation of thymidine kinase.

The PDX-1 positive human pancreatic ductal carcinoma cells can betargeted using a RIP-tk construct: It has also been discovered that aRIP-tk construct may be used to target PDX-1 positive human pancreaticductal carcinoma cells. As used herein, “PDX-1 positive” means cellsthat naturally contain or comprise, or are modified to contain orcomprise, a PDX-1 transcription factor.

The adult mammalian pancreas is composed of two distinct glands withdifferent functions: the islets and the exocrine glands. However, thereexists increasing evidence that these cell types originate from a singlecell early in embryonic development (St-Onge L, et al., 9(3) CURB. OPIN.GENET. DEV. 295-300 (1999)). PDX-1 both plays an important role in earlypancreatic development and in β-cell specific activation of the insulinpromoter in mature islets. (Ahlgren U, et al., 12(12) GENES DEV.1763-1768 (1998)).

Pancreatic ductal carcinoma resembles cells found early throughout theembryologic pancreas. Human ductal pancreatic adenomcarcinoma (PDA)cells are hypothesized to arise from a pluri-potential stem cell. It hasbeen determined that these cells contain the transcription factor PDX-1.Although PDX-1 plays an important role in embryonic pancreaticdevelopment, it is normally found only in the mature islets andactivates the insulin promoter. Following investigation, it wasdiscovered that human pancreatic ductal carcinoma cell lines could alsobe targeted both in vivo and in vitro using the rat insulin promoter(RIP) to drive the suicide gene thymidine kinase (tk). In addition, thetranscription factor (PDX-1) responsible for insulin promoter activationin these cells was identified.

Specifically, it was hypothesized that human pancreatic ductal carcinomacell lines retain their ability to produce the transcriptional machineryneeded to activate the insulin promoter. To confirm this, the ratinsulin promoter (RIP) was studied to determine if it could selectivelytarget human pancreatic ductal carcinoma cells for genetic manipulationin culture using the suicide gene thymidine kinase (tk) followed byganciclovir (GCV). Experiments were also undertaken to identify thetranscription factor(s) responsible for insulin promoter activation inthese cancer cells. The insulin promoter's activation is normallylimited to β-cells and a genetic construct containing the suicide genethymidine kinase driven by RIP should only effect dividing cells withthe transcriptional machinery available to activate the insulinpromoter.

The resulting data as shown in Examples 2-4, indicate that the RIP-tkgene is able to target PDX-1 positive human pancreatic ductal carcinomacells (both PANC-1 and CAPAN-1 cells), in vitro and in vivo, andfurther, that cell-specific cytotoxicity of human pancreatic ductalcarcinoma cells can be achieved using a RIP thymidine kinase constructand GCV in vitro and in vivo. The data also indicate that thetranscription factor PDX-1, important in early embryonic pancreaticdevelopment, is responsible for both the activation and the targeting ofthe rat insulin promoter in PDA cells. In addition, a liposomal genedelivery system was shown to be effective in vivo in scid mice.

In the development of the RIP-tk and RIP-LacZ genetic constructs, the0.502 kb that is useful for maximal transcription of a gene was used(Frazier M L, 880 ANNALS N.Y. ACAD. SCI. 1-4 (1999)). As noted, these502 base pairs contain six E box binding sites for the transcriptionfactor BETA-2 and three PDX-1 binding sites, of which only one containsthe most favorable PDX-1 binding sequence (CTTAAT). Both BETA-2 andPDX-1 are found and are responsible for insulin promoter activation inrodents and humans, however BETA-2 appears to play a dominant role inrodents while PDX-1 is dominant in humans (Frazier M L, 880 ANNALS N.Y.ACAD. SCI. 1-4 (1999)).

A number of cell types were used in the study. PANC-1, CAPAN-1, andMIA-1 cells are all human pancreatic ductal carcinoma cell lines derivedfrom three different humans (Lieber M, et al., 15(5) INT. J. CANCER741-7 (1975)). These cells form clinically relevant pancreatic ductalcell tumors when injected into the peritoneum of mice (Schwartz RE, etal., 126(3) SURGERY 562-567 (1999)). Human small cell lung carcinomacell line A549, and human breast carcinoma cell line (T47D) were alsochosen as control cancer cell lines because they represent a variety ofundifferentiated human tissues.

To determine whether these cell lines contained RIP activatingpromoters, RT-PCR was performed on whole RNA isolated from PANC-1,CAPAN-1, and MIA-1 cells looking for the presence of known RIPtranscription factors BETA-2 and PDX-1. BETA-2 was chosen because itappears to be the dominant transcription factor in rodents and thereforeresponsible for the majority of the activation of RIP in these animals(Naya F J, et al., 11 GENES DEV. 2323-2334 (1997)). The 502 base pairRIP used in the experiment contain six E box binding sites for BETA-2and three PDX-1 sites. Message for PDX-1 was found in PANC-1 and CAPAN-1cell lines but not in MIA-1. No message for BETA-2 was found in any ofthe cell types.

As noted, PDX-1 is responsible both for early embryonic pancreaticdevelopment and for insulin promoter activation in the mature islet.Normally, it is not found outside the β-cell once the pancreas matures(St-Onge L, et al., 9(3) CURB. OPIN. GENET. DEV. 295-300 (1999); AhlgrenU, et al., 12(12) GENES DEV. 1763-1768 (1998); Frazier M L, 880 ANNALSN.Y. ACAD. SCI. 1-4 (1999)). Interestingly, a message for PDX-1 wasdiscovered in the human pancreatic ductal carcinoma cell lines PANC-1and CAPAN-1, but not in MIA-1.

To further demonstrate that PDX-1 is responsible for RIP activation inPANC-1 and CAPAN-1 cells, nuclear extract was obtained and assayed tosee if any nuclear proteins bound to the PDX-1 binding site were foundon RIP. Once binding was established, an antibody specific for PDX-1 wasadded to identify if the PDX-1 protein is the nuclear protein bound toRIP. The resulting data confirmed that the transcription factor PDX-1 ispresent in PANC-1 and CAPAN-1 cells and binds to RIP.

It was then hypothesized that RIP-driven cytotoxic constructs would workin PDX-1 positive cancer cell lines. This was confirmed as follows.

Utilizing a RIP-driven marker gene, RIP-LacZ, only PANC-1 and CAPAN-1demonstrated LacZ gene expression in vitro, whereas the other cell linesdid not. All cell lines displayed LacZ gene expression using theubiquitous promoter LacZ construct (RSV-LacZ) (Table 1). These resultssupported the use of RIP as cell specific promoter to drive theexpression of genes in PDX-1 positive human pancreatic ductal carcinomacell lines.

The effectiveness of the cytotoxic gene construct, RIP-tk, was studiedin vitro and found to selectively kill PANC-1 and CAPAN-1 cells, but notthe other cell lines. No killing of PANC-1 and CAPAN-1 was seen with inuntransfected cells given GCV alone or with the use of a hollow vector.All cell lines demonstrated an increase in cell death with the RIP-tkgene governed by a ubiquitous promoter. These data demonstrate RIP-tkcell-specific cytotoxicity of PDX-1-positive, pancreatic cancer celllines in vitro.

To confirm that PDX-1 was responsible for the in vitro effects, thePDX-1 binding site on the RIP was mutated. There are three PDX-1 bindingsites, however the PDX-1 site (CTTAAT) was chosen because it is thedominant binding site and is close to the 3′ end of the promoter, makingit easier to mutate using PCR technology. LacZ gene expression in PANC-1and CAPAN-1 cell lines transfected with the mutated RIP-LacZ constructwas negligible compared to that seen with the unmutated RIP-LacZconstruct. These data indicate that PDX-1 is useful for RIP activationof gene expression in these cell lines.

More specifically, as shown in Example 2, only the pancreatic ductalcarcinoma cells PANC-1 turned blue after X-gal staining (p<0.05, n=32per cell type) and only PANC-1 and CAPAN-1 cells had detectable levelsof beta-galactosidase protein (p<0.05, n=16). A significant increase incell death was observed in PANC-1 and CAPAN-1 cells transfected withRIP-tk, while no significant increase in cell death was observed in A549or MIA-1 cells transfected with RIP-tk (p<0.05, n=32). PANC-1 andCAPAN-1 cells contained RNA for PDX-1, but not for BETA-2. MIA-1 cellsdid not contain RNA for either PDX-1 or BETA-2. A super shift wasobserved with the PDX-1 antibody and nuclear extract from both PANC-1and CAPAN-1. Decreased levels of beta-galactosidase protein was found inPANC-1 and CAPAN-1 cells transfected with the mutated RIP-LacZ gene whencompared to the wild type RIP-LacZ gene (p<0.05, n=8).

The effectiveness of RIP-LacZ and RIP-tk gene constructs against PANC-1was then studied in vivo using a scid mouse model. Scid mice areimmunodeficient and will not reject a human pancreatic cancer cell line.PANC-1 cells were injected intraperitoneally and tumors were visible byday 24. Once the model was established in the laboratory, the mice weretreated with the gene therapy beginning at day 24 post-tumor-injection.A liposomal gene-delivery system was chosen for simplicity andeffectiveness (Schwarz R E, et al., 126(3) SURGERY 562-567 (1999);Smyth-Templeton N, et al., 15 NATURE BIOTECHNOL. 647-652 (1997)). Theliposomal gene construct was delivered intraperitoneally and mice weregiven GCV intraperitoneally twice per day for 14 days. Followingtreatment, mice were observed for 60 days and sacrificed.

At necropsy, all nine mice treated with the gene therapy/GCV had novisible pancreatic tumors and one of nine had microscopic tumors on theliver. All control groups had large tumors. These data confirm thathuman PANC-1 cells can be selectively killed using systemic delivery ofRIP-tk/GCV gene therapy.

The resulting data supports the hypothesis that pancreatic ductalcarcinoma represents a de-differentiated cell found early inembryological development containing properties found both in theexocrine pancreas and in the mature islet and therefore able to activatethe insulin promoter. The data also demonstrates that RIP can be used totarget certain human pancreatic ductal carcinoma cell lines in vivo andin vitro. The data demonstrates that the RIP-tk gene may be used withGCV to target and kill these cells. The data also indicates that thetranscription factor PDX-1, important in early embryonic pancreaticdevelopment, is responsible for the activation of the rat insulinpromoter in these cells.

Example 3, further corroborates that human ductal pancreaticadenocarcinoma (PDA) cells that contain the transcriptional machinery toactivate RIP (e.g., PDX-1 positive) can be targeted with a RIP-tk gene.PDA cell lines CAPAN-1 (C-1) and MIA-1 (M-1) were evaluated to determineif they could be targeted using the rat insulin promoter (RIP) drivingthe thymidine kinase gene (tk). In addition, the transcription factorsknown to activate RIP in rodents (PDX-l or BETA-2) were evaluated todetermine which were responsible for RIP activation in human PDA cells.

As shown in Example 3, the resulting data provide further confirmationthat RIP can drive the expression of a gene in human PDA cells(CAPAN-1), and that the transcription factor PDX-1 is useful forpromoter activation in human PDA cells.

In sum, PDX-1 positive human pancreatic cancer-specific cytotoxicity wasachieved both in vivo and in vitro using RIP to drive the suicide gene,thymidine kinase. RIP activation of these cell lines was shown to beregulated by the transcription factor, PDX-1.

Alterations in insulin secretion in the SSTR-5 knock out mouse using theisolated perfused mouse pancreas model: In addition, alterations ininsulin secretion in the somatostatin subtype receptor 5 knock out mouseusing the isolated perfused mouse pancreas model were studied. Theinhibitory biological activities of somatostatin are mediated by fivehigh affinity receptors that all have been identified in the islets ofLangerhan. (Kumar U, et al., 48(1) DLABETES 77-85 (1999)). In mouseisolated islets, a SSTR-5 specific agonist inhibited insulin secretion,implicating SSTR-5's role in insulin homeostasis. (Fagen S P, et al.,124(2) SURGERY 254-258 (1998)). Recently, SSTR-2 has been shown to beinvolved in glucagon inhibition. (Strowski M Z, et al., 141(1)ENDOCRINOL. 111-7 (2000)).

The mouse SSTR-5 gene was cloned and ablated by homologous recombinationto further elucidate its role in insulin homeostasis. Whole pancreata ofyoung (three-month-old) and old (twelve-month-old) mice were isolatedand perfused to determine the effect glucose stimulation has on insulinsecretion in mice lacking the SSTR-5 gene over time.

As shown in Example 8, histological sections of islets suggest there isno difference in islet cell morphology between 3-month-old KO and3-month-old wt mice or 12-month-old KO and 12-month-old wt mice. Therewere no differences in weight between KO and aged match controls. Therewere no differences in basal insulin secretion between any of the mice.Additionally, glucose stimulation caused in a significant increase ininsulin secretion compared to basal in all mice. Three-month-old KO micedemonstrated a blunted first phase that was significant compared to allother mice (Table 8 and FIG. 7 ). Twelve-month-old KO mice demonstrateda significant augmentation of both first phase and second phase comparedto all other groups (Table 8 and FIG. 7).

As such, the phenotype of the SSTR-5 KO mouse appears to be a bluntedfirst phase in 3-month-old mice and an augmentation of glucosestimulated insulin secretion in 12-month-old mice. The data suggest thatthe SSTR-5 gene is involved in the regulation of glucose stimulatedinsulin secretion and has a limited role in basal insulin secretion. Thedata suggests that the genotypic loss of the SSTR-5 gene is partiallycompensated for in mice at three months of age. However, as the mice ageto 12 months these compensatory mechanisms are no longer functional andan augmentation of glucose stimulated insulin secretion is seen. Itmaybe concluded that SSTR-5 gene regulates glucose stimulated insulinsecretion and alterations of insulin release occur in the perfusedpancreata of both 3-month-old and 12-month-old SSTR-5 geneticallyablated mice.

Accordingly, one embodiment of the invention is directed to a method forselectively expressing a target gene in a pancreatic cell. This methodcomprises the step of delivering to the cell an effective amount of anagent containing a recombinant nucleic acid sequence, the sequencecomprising an insulin promoter operatively linked to the target gene.Although insulin promoters from various species of animals, includingman, may be used in the practice of the invention, in a preferredembodiment, the insulin promoter is a rat insulin promoter.

The agent may be delivered to the cell using any suitable means, such asby cotransfection. In one embodiment, the agent is delivered byinfecting with a recombinant viral vector, such as a recombinantadenovirus. For example, a gutless adenovirus may be prepared per theprotocol of Hardy et al. (Smyth-Templeton N, et al., 15 NATUREBIOTECHNOL. 647-652 (1997); Hardy S, et al., 71(3) J. VIROL. 1842-1849(1997)), or by using the gutless adenovirus available from the ShellCenter for Gene Therapy, Baylor College of Medicine (Houston, Tex.).

Alternately, a liposomal gene delivery system may be used. For example,liposomal complexes, such as those available from the Shell Center forGene Therapy, Baylor College of Medicine, may be used. Alternately,liposomal complexes may be prepared per the protocols of Schwarz R E, etal., 126(3) SURGERY 562-567 (1999), and Smyth-Templeton N, et al., 15NATURE BIOTECHNOL. 647-652 (1997). Liposome gene constructs may bedelivered using any suitable route of delivery, but in a preferredembodiment are delivered intraperitoneally.

The pancreatic cell is preferably one that expresses one or moretranscription factors selected from the group consisting of BETA-2,GATA4, E47 and PDX-1. If desired, the method may further comprisecotransfecting the cell with one or more insulin promoter transcriptionfactors selected from the group consisting of BETA-2, GATA4, E47 andPDX-1.

Pancreatic cells that may be targeted include PDX-1 positive pancreaticductal carcinoma cells, β-cell tumor cells, insulinoma cells, andβ-cells. Certain cell lines useful for research purposes may also beeffectively targeted, including PANC-1 cells, CAPAN-1 cells, and NIT-1cells.

The insulin promoter may be used to drive expression of any desiredgene. However, in a preferred embodiment, the promoter is functionallylinked to and drives expression of a gene encoding thymidine kinase. Inthis embodiment, the method further comprises the step of delivering tothe cell GCV, acyclovir, FIAU or 6-methoxypurine arabinoside in anamount effective to ablate the cell.

Another embodiment of the invention is directed to a method forselectively ablating pancreatic cells in a patient comprisingadministering to the patient an effective amount of an agent comprisinga recombinant nucleic acid sequence comprising a cytotoxic geneoperatively linked to an insulin promoter.

In a preferred method, the cytotoxic gene is the nucleic acid encodingthymidine kinase, and the method further comprises the step ofadministering GCV, acyclovir, FIAU or 6-methoxypurine arabinoside to thepatient in an amount effective to ablate the cells.

The present invention is not limited to thymidine kinase as thecytotoxic gene. As will be clear to those of skill in the art, variouscytotoxic genes may be used. For example, the cytotoxic gene may be adirectly cytotoxic gene, such as the gene encoding diphtheria toxin, thegene encoding ricin or the gene encoding caspase. Caspase is a geneproduct that promotes cell death by apoptosis. Alternately, thecytotoxic gene may be a suicide gene, such as the aforementioned geneencoding thymidine kinase. Suicide genes can make targeted cellssusceptible to specific drugs. Administering the drug to cells carryingsuch suicide genes results in cell death. For example, cells expressingthe thymidine gene are killed following treatment with GCV or a similardrug, whereas cells not expressing the thymidine kinase gene areunharmed by GCV treatment.

Preferably, the pancreatic cells being ablated express one or moretranscription factors selected from the group consisting of BETA-2,GATA4, E47 and PDX-1, and the promoter is a rat insulin promoter.

Another embodiment of the invention is directed to a method forselectively ablating a target in an individual using a suicide genecoupled to a promoter that is unique to the target. The method comprisestransfecting the individual with an agent comprising a recombinantnucleic acid sequence comprising a thymidine kinase gene operativelylinked or coupled to an insulin promoter, such as the rat insulinpromoter, followed by treating or administering to the individual aneffective amount of GCV, acyclovir, FIAU or 6-methoxypurine arabinosidein an amount sufficient to cause ablation of the target.

The cytotoxic effect may be enhanced by any mechanism that results inupregulating transcription of RIP-tk, such as, for example, addition offactors that upregulate transcription of RIP-tk or ablation of factorsthat inhibit transcription of RIP-tk. Factors that upregulatetranscription of the rat insulin promoter include, for example, theBETA-2, GATA4, PDX-1 and E47 transcription factors. Factors that inhibittranscription of the rat insulin promoter include factors in thesomatostatin signal transduction pathway, such as, for example, thesomatostatin receptor subtype-5 (SSTR-5). In a preferred embodiment, thecytotoxic effect may be enhanced by co-transfecting RIP-tk with at leastone of the rat insulin promoter transcription factors, BETA-2, GATA4,PDX-1 and E47.

In a preferred embodiment, liposomes are used for the delivery vehicle.Alternately, other delivery vehicles known to those of skill in the art,such as adenoviral vectors, may be used. Preferably, the targetcomprises cells that express one or more transcription factors selectedfrom the group consisting BETA-2, GATA4, E47 and PDX-1. For example, thetarget may comprise cells of pancreatic origin, including, pancreaticcancer, insulin secreting tumors, PDX-1 positive pancreatic ductalcarcinoma, β-cell tumors, insulinomas, 13-cells, PANC-1 cells, CAPAN-1cells, or NIT-1 cells.

Still another embodiment is directed to a method for treating ametabolic disease in an individual comprising administering to theindividual an effective amount of an agent comprising a recombinantnucleic acid sequence. The nucleic acid sequence comprises a cytotoxicgene operatively linked to an insulin promoter. Preferably, thecytotoxic gene encodes thymidine kinase and the method further comprisesthe step of administering to the individual an agent selected from thegroup consisting of GCV, acyclovir, FIAU and 6-methoxypurinearabinoside. The metabolic disease may be hypoglycemia orhyperinsulinemia, such as hypoglycemia due to an insulin-secreting tumoror cell. In the latter case, the method preferably comprises the step ofadministering GCV to the individual in an amount effective to ablate theinsulin-secreting tumor or cell.

The invention is also directed to compositions for selectively causingregression or ablation of a pancreatic cell. Such compositionspreferably comprise a recombinant nucleic acid sequence comprising atarget gene, such as the thymidine kinase gene, functionally oroperatively linked to an insulin promoter. In a preferred embodiment,the promoter is a rat insulin promoter. The composition may be containedin a liposome delivery system, such as those described above.Alternately, it may be contained in a viral vector. Cells which may beselectively targeted by the composition include the various cell linesdisclosed herein, including, but not limited to, PDX-1 positivepancreatic ductal carcinoma cells, β-cell tumor cells, insulinoma cells,and β-cells.

The present invention is not limited to the RIP-tk construct disclosedherein. The present invention is also broadly directed to methods andconstructs for targeting other tissues using unique promoters or genesspecific to a particular tissue, to deliver a cytotoxic or suicide gene,such as tk, to the tissue to then kill the tumor. For example, in oneembodiment of the invention, breast tissue may targeted by linking asuicide gene or other cytotoxin with an estrogen receptor promoterand/or a casein promoter. In another embodiment, liver tissue may betargeted by using the a fetal protein promoter.

Still another embodiment of the invention is directed towards a methodfor the production of a protein in a cell. The method comprisesdelivering a nucleic acid molecule to the cell, wherein the nucleic acidmolecule comprises an insulin promoter operatively linked to astructural nucleic acid sequence encoding the protein, and the cellcomprises the PDX-1 transcription factor after delivery of the nucleicacid molecule. Alternatively, the cell may comprise the BETA-2transcription factor after delivery of the nucleic acid molecule. Thecell may comprise the PDX-1 transcription factor or the BETA-2transcription factor before delivery of the nucleic acid molecule. Thecell may naturally comprise the PDX-1 transcription factor or the BETA-2transcription factor. The cell may generally be any type of cell, suchas a pancreatic cell. When the cell is a pancreatic cell, the protein ispreferably produced in the pancreatic cell at a higher concentrationthan in a non-pancreatic cell containing the nucleic acid molecule. Thecell may be a pancreatic ductal carcinoma cell, a β-cell tumor cell, aninsulinoma cell, or β-cell, and preferably is a pancreatic ductalcarcinoma cell. The cell may be PANC-1, CAPAN-1, or NIT-1 cell lines.

The insulin promoter may generally be any insulin promoter, preferablyis a rat insulin promoter, and more preferably is SEQ ID NO: 1.Alternatively, the insulin promoter can be an effective fragment of SEQID NO: 1. An effective fragment is a truncation of SEQ ID NO: I whichexhibits substantially the same transcription ability as does SEQ IDNO: 1. The effective fragment preferably exhibits at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, and ideally 100% of the transcriptionability of SEQ ID NO: 1. The effective fragment is preferably at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the lengthof SEQ ID NO: 1. Alternatively, the insulin promoter can be a promoterhaving a high level of percent sequence identity to SEQ ID NO: 1. Thelevel of percent sequence identity is preferably at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity as compared toSEQ ID NO: 1. Percent sequence identity is determined by aligning thetwo sequences with a commercial software package such as CLUSTALWversion 1.6 (Thompson, J. D., et al. Nucleic Acids Res. 22(22):4673-4680(1994)). The number of matches between the two aligned sequences isdivided by 502 and multiplied by 100 in order to obtain a percentsequence identity.

The cell may further comprise one or more transcription factors selectedfrom the group consisting of the BETA-2 transcription factor, the GATA4transcription factor, and the E47 transcription factor. The cell maycomprise both the PDX-1 transcription factor and the BETA-2transcription factor. The cell may comprise the PDX-1 transcriptionfactor, the BETA-2 transcription factor, and one or more transcriptionfactors selected from the group consisting of the GATA4 transcriptionfactor and the E47 transcription factor. The protein may generally beany protein, and preferably is thymidine kinase. The delivery step maycomprise contacting the cell with a recombinant viral vector.Alternatively, the delivery step may comprise contacting the cell withan adenovirus. The molecule may further comprise a structural nucleicacid sequence encoding one or more transcription factors selected fromthe group consisting of the BETA-2 transcription factor, the GATA4transcription factor, the E47 transcription factor, and the PDX-Itranscription factor.

The method may further comprise delivering to the cell one or moretranscription factors selected from the group consisting of the BETA-2transcription factor, the GATA4 transcription factor, the E47transcription factor, and the PDX-1 transcription factor. The method mayfurther comprise delivering a second nucleic acid molecule to the cell,wherein the second nucleic acid molecule encodes one or moretranscription factors selected from the group consisting of the BETA-2transcription factor, the GATA4 transcription factor, the E47transcription factor, and the PDX-1 transcription factor. The method mayfurther comprise delivering ganciclovir, acyclovir, FIAU, or6-methoxypurine arabinoside to the cell after delivery of the nucleicacid molecule. Delivering ganciclovir, acyclovir, FIAU, or6-methoxypurine arabinoside to the cell preferably ablates the cell. Anadditional embodiment of the invention is directed towards a method forablating cells in an individual comprising delivering an agent to theindividual, wherein the agent comprises a nucleic acid molecule, and thenucleic acid molecule comprises an insulin promoter operatively linkedto a structural nucleic acid sequence encoding a cytotoxic protein. Thecytotoxic protein can generally be any cytotoxic protein, and preferablyis thymidine kinase. The method may further comprise deliveringganciclovir, acyclovir, FIAU, or 6-methoxypurine arabinoside to theindividual after delivery of the agent. The insulin promoter maygenerally be any insulin promoter, preferably is a rat insulin promoter,and more preferably is SEQ ID NO: 1. Alternatively, the insulin promotermay be an effective fragment of SEQ ID NO: 1. An effective fragment is atruncation of SEQ ID NO: 1 which exhibits substantially the sametranscription ability as does SEQ ID NO: 1. The effective fragmentpreferably exhibits at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, and ideally 100% of the transcription ability of SEQ ID NO: I. Theeffective fragment is preferably at least about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% of the length of SEQ ID NO: 1.

Alternatively, the insulin promoter may be a promoter having a highlevel of percent sequence identity to SEQ ID NO: 1. The level of percentsequence identity is preferably at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity as compared to SEQ ID NO: 1. Thecells may further comprise one or more transcription factors selectedfrom the group consisting of the BETA-2 transcription factor, the GATA4transcription factor, and the E47 transcription factor. The cells maycomprise both the PDX-1 transcription factor and the BETA-2transcription factor. The cells may comprise the PDX-1 transcriptionfactor, the BETA-2 transcription factor, and one or more transcriptionfactors selected from the group consisting of the GATA4 transcriptionfactor and the E47 transcription factor. The molecule may furthercomprise a structural nucleic acid sequence encoding one or moretranscription factors selected from the group consisting of the BETA-2transcription factor, the GATA4 transcription factor, the E47transcription factor, and the PDX-1 transcription factor. The method mayfurther comprise delivering to the cell one or more transcriptionfactors selected from the group consisting of the BETA-2 transcriptionfactor, the GATA4 transcription factor, the E47 transcription factor,and the PDX-1 transcription factor. The method may further comprisedelivering ganciclovir, acyclovir, FIAU, or 6-methoxypurine arabinosideto the cell after delivery of the nucleic acid molecule. Deliveringganciclovir, acyclovir, FIAU, or 6-methoxypurine arabinoside to the cellpreferably ablates the cell. The cells may generally be any type ofcells. The cells may be pancreatic cells. The cells may be insulinsecreting cancer cells. The cells may be PDX-1 positive pancreaticductal carcinoma cells, pancreatic ductal carcinoma cells, β-cell tumorcells, insulinoma cells, or β-cells. The cells may be PANC-1 cells,CAPAN-1 cells, or NIT-1 cells. The agent may comprise liposomes oradenoviral vectors. The individual is preferably a mammal, and morepreferably is a human.

An additional embodiment of the invention is directed towards a methodof treating a metabolic disease in an individual, comprising deliveringan agent to the individual, wherein the agent comprises a nucleic acidmolecule, and the nucleic acid molecule comprises an insulin promoteroperatively linked to a structural nucleic acid sequence encoding acytotoxic protein. The metabolic disease may generally be any metabolicdisease, and preferably is hypoglycemia or hyperinsulinemia. Theindividual is preferably a mammal, and more preferably is a human. Thecytotoxic protein may generally be any cytotoxic protein, and preferablyis thymidine kinase. The method may further comprise deliveringganciclovir, acyclovir, FIAU, or 6-methoxypurine arabinoside to theindividual after delivery of the agent. The insulin promoter maygenerally be any insulin promoter, preferably is a rat insulin promoter,and more preferably is SEQ ID NO: 1. Alternatively, the insulin promotermay be an effective fragment of SEQ ID NO: 1. An effective fragment is atruncation of SEQ ID NO: 1 which exhibits substantially the sametranscription ability as does SEQ ID NO: 1. The effective fragmentpreferably exhibits at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, and ideally 100% of the transcription ability of SEQ ID NO: 1. Theeffective fragment is preferably at least about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% of the length of SEQ ID NO: 1. Alternatively,the insulin promoter may be a promoter having a high level of percentsequence identity to SEQ ID NO:1. The level of percent sequence identityis preferably at least about 80%, 85%,90%, 95%,96%,97%, 98%, or 99%sequence identity as compared to SEQ ID NO: 1. The metabolic disease maycomprise an insulin-secreting tumor or an insulin-secreting cell.

A further embodiment of the invention is directed towards an isolatednucleic acid molecule comprising an insulin promoter operatively linkedto a structural nucleic acid sequence encoding a cytotoxic protein. Thecytotoxic protein may generally be any cytotoxic protein, and preferablyis thymidine kinase. The insulin promoter may generally be any insulinpromoter, preferably is a rat insulin promoter, and more preferably isSEQ ID NO: 1. Alternatively, the insulin promoter may be an effectivefragment of SEQ ID NO: 1. An effective fragment is a truncation of SEQID NO: 1 which exhibits substantially the same transcription ability asdoes SEQ ID NO:1. The effective fragment preferably exhibits at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and ideally 100% of thetranscription ability of SEQ ID NO: 1. The effective fragment ispreferably at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% of the length of SEQ ID NO: 1.

Alternatively, the insulin promoter may be a promoter having a highlevel of percent sequence identity to SEQ ID NO: 1. The level of percentsequence identity is preferably at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity as compared to SEQ ID NO: 1. Thenucleic acid molecule may comprise a viral vector.

An additional embodiment of the invention is directed towards a kitcomprising an isolated nucleic acid molecule, wherein the isolatednucleic acid molecule comprises an insulin promoter operatively linkedto a structural nucleic acid sequence encoding a cytotoxic protein. Theisolated nucleic acid molecule may be any of the isolated nucleic acidmolecules described herein. The nucleic acid molecule may be containedin a container. The kit may further comprise one or more agents selectedfrom the group consisting of ganciclovir, acyclovir, FIAU, and6-methoxypurine arabinoside. The kit may comprise ganciclovir. The agentmay be contained in a second container. Alternatively, the nucleic acidmolecule and the agent may be contained in the same container. Thecytotoxic protein may generally be any cytotoxic protein, and preferablyis thymidine kinase. The cytotoxic protein is preferably cytotoxic tomammals, and more preferably is cytotoxic to humans.

A further alternative embodiment of the invention is directed towards amethod for increasing the secretion of insulin in an individualcomprising reducing the concentration of somatostatin receptor. Thereducing step may comprise adding an agent which knocks out the nucleicacid sequence encoding the somatostatin receptor. The agent may be anucleic acid molecule. The reducing step may comprise adding an antibodyto the individual which binds to the somatostatin receptor. Theindividual preferably is a mammal, and more preferably is a human. Thesomatostatin receptor preferably is somatostatin subtype receptor 5. Thesomatostatin receptor preferably is in the pancreas of the individual.

The following examples are offered to illustrate embodiments of theinvention, and should not be viewed as limiting the scope of theinvention.

EXAMPLE 1 Demonstration of β-cell Specific Cytotoxicity Using a RatInsulin Promoter Thymidine Kinase Construct

Materials, methods and summary of results: 0.502 kb of RIP (SEQ IDNO: 1) was ligated to the reporter gene LacZ and ligated to tk. Thesetwo genes were transfected into several cell lines to ascertain β-cellspecific expression and β-cell specific cytotoxicity in vitro. RT-PCRand EMSA were performed on NIT-1 cell RNA and nuclear extract,respectively, to determine the transcription factors present andresponsible for RIP activation in NIT-1 cells. A mouse cell adenomamodel was created with NIT-1 cells. These mice were treated with theRIP-tk gene and both blood sugars and animal viability were monitored.

Only the beta (NIT-1) cells stained blue after X-gal staining (p<0.05,n=16) or had detectable levels of beta-galactosidase protein (p<0.05,n=6) in vitro. A significant decrease in cell survival was observed inNIT-1 cells transfected with RIP-tk, in vitro (p<0.05, n=48). MessengerRNA for both BETA-2 and PDX-1 was found in NIT-1 cells and a super shiftwas observed for both BETA-2 and PDX-1. NIT-1 cell tumors weresuccessfully targeted with the RIP-LacZ gene. Mice inoculated with NIT-1cells developed clinically relevant tumors with hypoglycemia and deathat sixty days post inoculation. The in vivo delivery of the RIP-tk genein combination with GCV inhibited hypoglycemia and animal death (n=30,p<0.05).

Generation of RIP-LacZ and RIP-tk constructs: All restriction enzymesunless otherwise noted were from GIBCO-BRL, Bethesda, Md. The plasmidpD46.21 (provided by Dr. Franco DeMayo, Departments of Cell Biology andPediatrics, Baylor College of Medicine), which contains aβ-galactosidase gene with a polyadenylation signal and a nuclearlocalization signal, was digested with HindIII, blunt ended with Klenow,and digested with BamHI. RIP in Blue-Script KS⁺ (provided by Dr.Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine)was isolated with SstII, blunt ended with T4 polymerase (Promega,Madison, Wis.), digested with BamHI and ligated into restrictionendonuclease treated pD46.21 following the standard procedure. Theconstruct was verified by digesting with Notl and identifying a 4.1 kbband.

The RIP-tk construction was generated in two steps. First the codingsequence of tk, 66 by upstream from the ATG, was isolated frompMC-1-TK-6 (provided by Dr. Arthur Beaudet, Department of Cell Biology,Baylor College of Medicine) with NotI, blunt ended with Klenow, anddigested with BamHI. The isolated tk gene was then ligated with growthhormnone polyA (GhpA), in Blue-Script, which was digested with EcoRI,blunt ended with Klenow, and digested with BamtHI. This ligation wasverified with HindIII.

Thymidine kinase-GHpA in Blue-Script was further digested with BamHI andNotI and ligated with isolated 0.502 kb of RIP previously digested withBamHI and NotI. The construct was verified by digesting with BamHI andNotI and identifying a 0.5 kb band.

Transient transfection of genetic constructs/transfection efficiency:All cell lines were obtained from The Tissue Core Facility at BaylorCollege of Medicine and grown in their respective media as recommendedby ATCC, Bethesda, Md. Cells were plated into six well dishes growing inlogarithmic phase (60-80% confluent) twenty-four hours prior totransfection. All cells were transfected with 1 g of DNA per well(RIP-LacZ, RSV-LacZ, RIP-tk, hollow vector, and MC-1-tk). The DNA wasmixed with 6:1 of Fugene (Boehringer Mannheim, Indianapolis, Ind.) in94:1 of Dulbecco's modified Eagle's medium without serum (Gibco-BRL,Bethesda, Md.). 100:1 of solution was added to each well by mixinggently.

The maximum transfection efficiency for NIT-1 cells in vitro was tenpercent. This was demonstrated using both the RIP-LacZ gene and theRSV-LacZ gene. Despite this transfection efficiency a 26% decrease incell survival was observed in NIT-1 cells transfected with the RIP-tkgene. This may be accounted for by the bystander effect. The bystandereffect results from the transfer of viral thymidine kinase proteins intocells through gap junctions (Pope IM, et al., 33(7) EUR. J. CANCER.1005-16 (1997)). This allows for cells that were not originallytransfected with a viral tk gene to become susceptible to thecytotoxicity of GCV, thereby increasing the number of cells beingkilled.

Detection of β-galactosidase gene expression, X-gal staining: NIT-1,CV-1 (monkey renal cell), F9 (mouse embryonic carcinoma cell), 3T3(mouse fibroblast cell) and H411 (mouse lung cell) cells weretransfected with either RIP-LacZ or RSV-LacZ (provided by Dr. JeffRosen, Department of Cell Biology, Baylor College of Medicine). RSV-LacZserved as a positive control to ensure that all cells were sufficientlytransfected. Thirty-six hours post transfection cells were stained withX-gal staining solution to detect for the presence of thebeta-galactosidase protein. This was done by first washing the cellswith cold PBS (twice) and fixing them with 0.5% glutaraldehyde for 5minutes. Cells were then washed with cold PBS (twice), and a X-galstaining solution containing IM Mg-CI₂, 5M NaCl, 0.5M HEPES, pH 7.3, 30mM potassium ferricyanide, 30 mM potassium ferrocyanide, and 2% X-galsolution was added. Cells were then incubated at 37° C. for 24 hrs todevelop the color. An independent observer verified the presence of bluecolor. The experiment was repeated four times and a total of sixteenwells examined per cell type.

Only the β-cell line NIT-1 demonstrated blue color, after transfectionwith RIP-LacZ (n=16, p<0.05). All cell lines transfected with RSV-iacZdeveloped blue color after X-gal staining ensuring that all cell typeswere adequately transfected. The percentage of NIT-1 cells staining bluewith the RSV-LacZ construct equaled the percentage of blue stainingcells with the RIP-LacZ construct and was assumed to be the transfectionefficiency for NIT-1 cells for this experiment.

Detection of β-galactosidase gene expression, using a luminometer: NIT-1and F9 cells transfected with either RIP-LacZ or RSV-LacZ were subjectedto beta-galactosidase reporter gene assay (Tropix, Bedford, Mass.). Theassay was carried out in triplicate. Protein levels were determinedusing Bradford's protein assay (Sigma, St. Louis, Mo.). Results arerepresented in light units and adjusted for protein content.

Transfection of RIP-LacZ resulted in a significant increase inbeta-galactosidase protein levels in NIT-1 cells compared to F9 cells,2.9×10⁵ light units for NIT-1 cells and 1.2×10⁵ light units for F9 cells(n=6, p<0.05). Both cell types demonstrated an equal amount ofbeta-galactosidase after transfection with RSV-LacZ, 3.7×10⁵ light unitsfor NIT-1 cells and 3.5×10⁵ light units for F9 cells (n=6, p=NS). Thisensured that F9 cells were adequately transfected. Background lightunits were equal between cell types as well, 1.3×10⁵ light units forNIT-1 cells and 1.4×10⁵ light units for F9 cells (n=6, p=NS).

Ganciclovir dose response curve for NIT-1 cells in culture: Doseresponse curves with GCV for untransfected NIT-1 and F9 (control) cellswere performned prior to cytotoxicity studies with genetic constructs.NIT-1 cells were plated into a ninety-six well plate at a density of5,000 cells per well and treated with GCV (0.1 to 2.5 μg/ml) todetermine a dose response curve to GCV alone. The cells were treateddaily for five days and cell viability was ascertained with an MTSassay. GCV was found to be toxic to untransfected NIT-1 cells at muchlower doses than has been reported in the literature for other celltypes (FIG. 1A) (Al-Hendy A, et al., 43 GYNECOL. OBSTET. INVESTIG.268-275 (1997); Eastham J, et al., 7 HUMAN GENE THER. 515-523 (1996);Chen S-H, et al., 91 PROC. NATL. ACAD. SCI. USA 3054-3057 (1994); TongX, et al., 61 GYNECOL. ONCOL. 175-179 (1996)). The GCV toxicity doseresponse curve for F9 cells resembled other cell lines (FIG. 1B).Specifically, FIG. 1A shows the dose response curve for NIT-1 cellsgiven GCV. The x-axis shows GCV in μg/ml. The y-axis shows percent cellsurvival. Note that at concentrations of GCV 0.25: g/ml and higher asignificant decrease in cell survival is observed, n=8, *p<0.05,Unpaired Student t-test. FIG. 1B shows the dose response curve for F9cells given GCV. The x-axis shows GCV in μg/ml. The y-axis shows percentcell survival. F9 cell toxicity resembles that of other published celllines, n=8,*p<0.05, unpaired Student t-test.

At GCV dosages greater than 0.25 μg/ml survival for untransfected NIT-1cells significantly decreased (n=8, p<0.05) (FIG. 1A). Based on thisdata, it was determined that 0.20 μg/ml is the maximum dose of GCV onecan use to treat NIT-1 cells in culture. This is believed to be relatedto the fastidious nature of NIT-1 cells in culture, because it is notreproducible in vivo.

Treatment of transfected cells with ganciclovir: NIT-1 and F9 cells weretransfected with either the RIP-tk gene or a hollow vector (V)(negativecontrol). A group of untransfected cells (C) was used to control for theeffect of GCV alone on cell death. As a positive control, F9 cells weretransfected with a tk construct driven by a ubiquitous promoter(MC-1-tk) to ensure that the F9 cells were being sufficientlytransfected and susceptible to the cytotoxic effects of thymidinekinase. Twenty-four hours post transfection the cells were re-platedinto ninety-six well plates at a density of 5,000 cells per well, andsubjected to GCV treatment with a dose of 0.5 to 0.20 μg/ml of GCV. Themedia was refreshed daily. Cell viability was determined by using an MTSassay (Promega, Madison, Wis.) read at an absorbency of 490 nm. Percentcell death was calculated utilizing the following formula:$\frac{A - B}{A} \times 100$where A is the absorbency at 490 nm of transfected cells not treatedwith GCV and B is the absorbency at 490 nm of transfected cells treatedwith GCV. Percent cell survival was calculated by subtracting thepercent cell death from one hundred.

In cells transfected with RIP-tk, only NIT-1 cells demonstrated asignificant and GCV dose dependent decrease in cell survival; 17.7% and26.8% for 0.15 μg/ml and 0.2 μg/ml of GCV, respectively.

Specifically, FIG. 2 is a bar graph which shows the percent cellsurvival of NIT-1 cells transfected with nothing (C), a hollow vector(V), and RIP-tk with increasing levels of GCV (μg/ml). As noted, onlythe RIP-tk gene demonstrated significant and GCV dose dependent decreasein NIT-1 cell survival, n=48, *p<0.05, ANOVA.

F9 cells failed to show any significant decrease in cell survival withthe R[β-tk gene following GCV treatment. F9 cells transfected with thepositive control MC-1-tk did demonstrate a significant decrease in cellsurvival; 15.5% and 24.3% for 0.15 μg/ml and 0.2 μg/ml of GCV,respectively (FIG. 3). Neither cell type demonstrated any significantdecrease in survival following transfection with a hollow vector (V) orwith GCV treatment alone (C). Specifically, FIG. 3 is a bar graph whichshows the percent cell survival of F9 cells transfected with nothing(C), a hollow vector (V), RIP-tk, and MC-1-tk with increasing levels ofGCV (μg/ml). As noted, no significant decrease in cell survival wasdemonstrated with the RIP-tk gene. A significant and GCV dose dependentdecrease in F9 cell survival was demonstrated with the MC-1-tk ensuringthat F9 cells were both adequately transfected and susceptible to thetoxic effects of tk with GCV (n=48, *p<0.05, ANOVA).

NIT-1 mRNA isolation and RT-PCR analysis of transcription factors PDX-1and BETA-2: NIT-1 total RNA was extracted using RNAzol™ (Tel-Test, INC.,Friendswood, Tex.). Briefly, cells were allowed to grow to confluence inlarge vented flasks. Media was removed and 5 ml of RNAzolT was added.The cells were removed with the aid of a cell scraper and placed on icefor fifteen minutes. 500 μl of chloroform was added and the cells werespun for fifteen minutes at 10,000 rpm at 4° C. The aqueous phase wasremoved and mixed with an equal volume of isopropanol and placed at 4°C. for fifteen minutes and then spun for fifteen minutes at 15,000 rpmat 4° C. The pellet was then washed with 70% ethanol and re-spun at15,000 rpm and allowed to dry.

Reverse transcriptase polymerase chain reaction (RT-PCR) was performedusing SUPERSCRIPTM Preamplification System for First Strand cDNASynthesis kit (Gibco-BRL, Bethesda, Md.). Primers specific for mousePDX-1 mRNA (forward 281-300 bps tgaacagtgaggagcagtac (SEQ ID NO: 5) andreverse 870-889 bps ttttccacttcatgcgacgg (SEQ ID NO: 6)) and primersspecific for both human and mouse BETA-2 (forward bps 944-964,cgccgagtttgaaaaaaatt (SEQ ID NO: 7) and reverse bps 1227-1207,tttttccgacggaagacatt (SEQ ID NO: 8)), (BETA-2 primers were provided byDr. Ming-Jer Tsai, Department of Cell Biology, Baylor College ofMedicine) were used. Standard β-actin primers were used as controls. PCRprogram for all three primers was as following: 2 min 94° C., thenthirty cycles of 1 min 94° C., 1 min 55° C., and 1 min 72° C. and 5 min72° C. for finishing. 5:1 of the PCR reaction was run on gelelectrophoresis. A 600 bp band identified a positive for PDX-1 and a 300bp band identified a positive for BETA-2. Thus, NIT-1 cells demonstrateda message for PDX-1 and BETA-2.

Nuclear extracts and electrophoretic mobility-shift assays (EMSA): NIT-1nuclear extracts were isolated as described elsewhere (Osaki T, et al.,54 CANCER RES. 5258-5261 (1994)). Protein levels were determined usingBradford's protein assay (Sigma, St. Louis, Mo.). A double-strandedoligodeoxynucleotide probe that contained both a BETA-2 site and a PDX-1site within RIP (bp 350-381 on RIP TTGGCCATCTGCTGATCCACCCTTAATGGGAC; SEQID NO: 9) was labeled with ∀³²P dGTP by filling overhanging 5′ ends withSuperscriptTM (Gibco-BRL, Bethesda, Md.). Binding reactions wereperformed with 2.5 μg of protein and 1 μl hot probe per lane on a 5%acrylamide gel with either nuclear extract alone or 10 OX cold wild typeprobe to assess protein binding. Supershift analysis for BETA-2 andPDX-1 binding activity was performed by the addition of 1:1 of antiBETA-2 antibody (provided by Dr. Ming-Jer Tsai, Department of CellBiology, Baylor College of Medicine) and 1:1 of anti-N-terminal XIHbox8antibody (provided by Dr. Christopher Wright, Department of Medicine,Vanderbilt School of Medicine), respectively.

Nuclear extract from NIT-1 cells demonstrated binding to the labeledoligonucleotide in RIP that contained both the BETA-2 and PDX-1 sites.Furthermnore a supershift was observed with both the BETA-2 and PDX-1antibodies.

Creation of an in vivo mouse β-cell adenoma model: Twelve femaleICR/scid mice, age 6-10 weeks underwent intraperitoneal injections of5×10⁶ NIT-1 cells as described (Tong X, et al., 61 GYNECOL. ONCOL.175-179 (1996)). An additional twelve female ICR/scid mice, age 6-10weeks underwent an equal volume of PBS and were used as controls. Toestimate tumor growth blood glucose was measured from the tail veinusing a glucometer every other day. At animal death the mice underwentnecrotopsies.

ICR/scid mice injected EP with 5×10⁶ NIT-1 cells initially demonstratedno difference in blood sugars as compared to control mice. However,eight days prior to their death the mice demonstrated a rapid decline oftheir blood glucose levels and died (see FIG. 4). At autopsy, with theexception of one mouse, which had a small (<0.2mm) subcutaneous tumor,no tumors were identified. NIT-1 cells added with PBS, GCV, or RIP-LacZand GCV led to the described rapid decline in blood glucose levels.Addition of NIT-1 cells with RIP-tk and GCV prevented the decrease inblood glucose levels.

Treatment of β-cell adenomas (insulinomas) iln vivo: twenty-seven femaleICRiscid mice, age 6-10 weeks old underwent intraperitoneal injectionsof 5×10⁶ NIT-1 cells and nine female ICR/scid mice, age 6-10 weeks oldunderwent intraperitoneal injections of PBS. At day 31, the mice wererandomized to receive either the RIP-tk gene, the RIP-LacZ gene, ornothing. The RIP-LacZ construct served both as a control and to localizegene expression. The genes were delivered via liposomes IP (liposomesprovided by Dr. Nancy Smyth-Templeton Center for Gene Therapy andDepartment of Cell Biology, Baylor College of Medicine) and mixed asdescribed (Smyth-Templeton N, et al., 15 NATURE BIOTECHNOL. 647-652(1997)). Six mice were randomized to receive the RIP-tk gene followed byseven days of GCV (40mg/kg IP). Six mice were randomized to receive theRIP-LacZ gene followed by seven days of GCV (40mg/kg IP). Six mice wererandomized to receive PBS followed by seven days of GCV (40mg/kg IP).Six mice that were inoculated with NIT-1 cells were randomized toreceive seven days of PBS. Six mice which were inoculated with PBS wererandomized to receive the RIP-tk gene followed by seven days of GCV(40mg/kg IP).

Three mice that were inoculated with NIT-1 cells and three mice thatwere inoculated with PBS were randomized to receive the RIP-LacZ geneand then sacrificed thirty-six hours later. These mice had their tissuesfixed including brain, heart, lung, liver, small bowel, spleen, kidneys,and pancreas in 5% glutaraldehyde for one hour, were stained with X-galstaining solution for twenty-four hours, and were sectioned and counterstained with nuclear fast red. Analysis of treatment and gene placementconsisted of normalization of blood glucose values, inhibition of animaldeath, and presence of blue nuclei after X-gal staining.

NIT-1 cell tumors were successfully targeted with the RIP-LacZ gene withtumors staining blue after fixation and X-gal staining. Five out of sixexperimental mice (received NIT-1 tumors, RIP-tk gene, and GCV) livedthroughout the length of the experiment without any significant changein blood glucose values. The mouse that did demonstrate a decline ofblood glucose levels and death had a technical problem in itsadministration of the RIP-tk gene; it was injected into the bladder. Allof the other mice that received NIT-1 cells underwent a rapid decline inblood glucose values and died within sixty days of tumor inoculation(see FIG. 4).

ICR/scid mice injected with 5×10⁶ NIT-1 cells exhibited an interestingresponse to increasing tumor burden. Despite the mice dying at differenttime points they all exhibited the same sequence of blood sugars priorto death; eight days prior. to death they underwent a sequentialdecrease in blood sugars. This indicates there is a critical tumorburden that the mice can tolerate before their regulatory mechanisms areovercome and the mice die rapidly from hypoglycemia. The time it took toreach this critical tumor volume was not the same for each mouse andthis can be explained, in part, by a learning curve associated withinjecting the mice IP. Some mice probably had more tumor escape throughthe needle tract than others. In fact, one mouse had implantation of thetumor into the subcutaneous space. This proved to be beneficial becauseno tumors were discovered in the peritoneum of the other mice. In thesubsequent in vivo experiment there were no subcutaneous tumors, and allof the mice began their decline in blood sugars and died within one weekof each other, indicating that the technique had improved.

RIP driven expression in NIT-1 cells in vivo: In another murinehypoglycemic tumor model, 5×10⁶ NIT-1 cells (or PBS) were injectedintraperitoneally (IP) into ICR/scid mice (n=12 per group). Asignificant reduction of blood glucose values at 53±7 days and death at60±7 days was observed in mice that received the NIT-1 cells. In asecond experiment additional mice were randomized (n=6 per group) asfollows: 1) NIT-1, RIP-tk and ganciclovir (GCV); 2) NIT-1, RlIP-LacZ andGCV; 3) NIT-1 and GCV only; and 4) NIT-1 and PBS only. GCV was given at40 mg/kg IP BID for 7 days. RIP-tk and RIP-LacZ genetic constructs weregenerated in the laboratory and were delivered EP 31 days post tumorinjection by complexing the DNA with 20 mM of extrudedDOTAP:cholesterol. Other mice injected with tumors and RIP-LacZ hadtheir tissues stained with X-gal. Blood glucose values, animal survival,and blue cells after X-gal staining were recorded. RT-PCR was done onNIT-1 RNA with primers specific for BETA-2 and PDX-1. A band shift wasperformed by mixing NIT-1 nuclear extract with RIP and antibodiesspecific to PDX-1 and BETA-2.

At animal death the insulinoma cells were identified by X-gal staining.RIP-tk in combination with GCV inhibited hypoglycemia and animal deathin all mice (see FIG. 4). BETA-2 and PDX-1 RNA was found in NIT-1 cellsand both transcription factors demonstrated RIP binding with a supershift on the band shift.

Statistics: X-gal staining of cells used in this example (NIT-1, CV-1,F9, 3T3, and H411) were compared using both ANOVA and chi square test.The light units were adjusted for protein content and compared using anunpaired Student's t-test. Cell survival between different constructsand cell lines was compared with ANOVA. Mouse blood glucose levels werecompared with ANOVA and animal survival was compared by chi square test.P <0.05 represented significance.

EXAMPLE 2 Human Pancreatic Ductal Carcinoma Cells can be Targeted Usinga RIP-Tk Construct in Vitro

Materials, methods and summary of results: 0.502 kb of RIP was ligatedto the reporter gene LacZ and transfected into several human cell lines:human pancreatic ductal carcinoma cell lines (PANC-1, CAPAN-1, andMIA-1), lung carcinoma (A549), and breast carcinoma (T47D). X-galstaining and the detection of beta-galactosidase using a luminometeranalyzed LacZ gene expression. RIP was ligated to tk and transfectedinto PANC-1, CAPAN-1, MIA-1, and A549 cells. Cell viability was comparedafter transfection with the RIP-tk genetic construct and daily treatmentwith of ganciclovir (GCV). RT-PCR was performed on PANC-1, CAPAN-1, andMIA-1 total RNA with primers specific for known insulin transcriptionfactors PDX-1 and BETA-2. EMSA was also performed on PANG-1 and CAPAN-1nuclear extract using an antibody specific to PDX-1. A mutated RIP-LacZconstruct was created with one PDX-1 binding site changed. Thisconstruct was transfected into PANC-1 and CAPAN-1 cells and the amountof beta-galactosidase protein using a luminometer was assessed.

Only the pancreatic ductal carcinoma cells PANC-1 turned blue afterX-gal staining (p<0.05, n=32 per cell type) and only PANC-1 and CAPAN-1cells had detectable levels of beta-galactosidase protein (p<0.05,n=16). A significant increase in cell death was observed in PANC-1 andCAPAN-1 cells transfected with RIP-tk, while no significant increase incell death was observed in A549 or MIA-1 cells transfected with RIP-tk(P<0.05, n=32). PANC-1 and CAPAN-1 cells contained RNA for PDX-1, butnot for BETA-2. MIA-1 cells did not contain RNA for either PDX-1 orBETA-2. A super shift was observed with the PDX-1 antibody and nuclearextract from both PANC-1 and CAPAN-1. Decreased levels ofbeta-galactosidase protein was found in PANC-1 and CAPAN-1 cellstransfected with the mutated RIP-LacZ gene when compared to the wildtype RIP-LacZ gene (p<0.05, n=8). Finally, RIP was successfully used ina mouse model to drive expression of LacZ and tk in PANC-1 cells invivo.

The data show that the RIP-tk gene is able to target and kill bothPANC-1 and CAPAN-1 cells. The data also suggest that the transcriptionfactor PDX-1, important in early embryonic pancreatic development, isresponsible for both the activation and the targeting of the rat insulinpromoter in PANC-1 and CAPAN-1 cells.

Generation of RIP-LacZ and RIP-tk constructs: All restriction enzymesunless otherwise noted were from GIBCO-BRL, Bethesda, Md. The plasmidpD46.21 (provided by Dr. Franco DeMayo, Departments of Cell Biology andPediatrics, Baylor College of Medicine), which contains aβ-galactosidase gene with a polyadenylation signal and a nuclearlocalization signal, was digested with HindIII, blunt ended with Kienow,and digested with BamHIl. RIP in Blue-Script KS⁺ (Stratagene, La Jolla,Calif.) (provided by Dr. Ming-Jer Tsai, Department of Cell Biology,Baylor College of Medicine) was isolated with SstII, blunt ended with T4polymerase (Promega, Madison, Wis.), digested with BamlI and ligatedinto restriction endonuclease treated pD46.21 following the standardprocedure. The construct was verified by digesting with Not] andidentifying a 4.1 kb band.

The RIP-tk construction was generated in two steps. First the codingsequence of tk, 66 by upstream from the ATG, was isolated frompMC-1-TK-6 (provided by Dr. Arthur Beaudet, Department of Cell Biology,Baylor College of Medicine) with NotI, blunt ended with Klenow, anddigested with BamHI. The isolated tk gene was then ligated with growthhormnone polyA (GhpA), in Blue-Script, which was digested with EcoRI,blunt ended with Klenow, and digested with BamHI. This ligation wasverified with HindIII.

Thymidine kinase-GHpA in Blue-Script was further digested with BamHI andNoti and ligated with isolated 0.502 kb of RIP previously digested withBamHI and NotI. The construct was verified by digesting with BamHI andNotI and identifying a 0.5 kb band.

Transient transfection of genetic constructs: All PANC-1, CAPAN-1,MIA-1, A549 and T47D cell lines were obtained from American Tissue CoreFacility (ATCC) (Bethesda, Md.). Cells were plated into six well dishesgrowing in logarithmic phase (60-80% confluent) twenty-four hours priorto transfection. All cells were transfected with 3 μg of DNA per well(RIP-LacZ, RSV-LacZ, RIP-tk, hollow vector, and U-tk). The DNA was mixedwith 6 μl of Fugene (Boehringer Mannheim, Indianapolis, IN) in 94:1 ofDulbecco's modified Eagle's medium without serum (Gibco-BRL, Bethesda,Md.). 100:1 of solution was added to each well by mixing gently.

Detection of β-galactosidase gene expression, X-gal staining: PANC-1,CAPAN-1, MIA-1, A549, and T47D cells were transfected with eitherRIP-LacZ or RSV-LacZ (provided by Dr. Jeff Rosen, Department of CellBiology, Baylor College of Medicine). RSV-LacZ served as a positivecontrol to ensure that all cells were sufficiently transfected.Thirty-six hours post transfection, cells were stained with X-galstaining solution by first washing the cells with cold PBS (twice) andfixing them with 0.5% glutaraldehyde for 5 minutes. Cells were thenwashed again with cold PBS (twice), and a X-gal staining solution(containing 1M MgCl₂, 5M NaCl, 0.5M HEPES, pH 7.3, 30 mM potassiumferricyanide, and 2% X-gal solution) was added. Cells were thenincubated at 37° C. for 6-24 hrs to develop the color.

As shown in Table 1, despite the variety of cell types, only the humanpancreatic ductal carcinoma cell line PANC-1 demonstrated anysignificant blue color after transfection with RIP-LacZ (n=32, p<0.05).TABLE 1 Cell Type PANC-1 CAPAN-1 MIA-1 A549 T47D RIP-LacZ 8%^(Δ) 0% 0%0% <0.01% expression RSV-LacZ 8%* 0% 8%^(T) 9%^(T)    6%^(T) expression

Table 1: Representation of LacZ expression after transfection withRIP-LacZ or RSV-LacZ and X-gal staining. Results verified by anindependent observer and recorded as either positive or negative forblue color. Note that neither the RIP-LacZ nor the RSV-LacZ demonstratedany staining for the CAPAN-1 cells. N=32 for each cell type, ^(Δ)p<0.05,PANC-1 with RIP-LacZ vs MIA-1, A549, and T47D with RIP-LacZ, ANOVA.*p=NS, PANC-1 with RSV-LacZ vs MIA-1, A549, and T47D with RSV-LacZ,ANOVA. ^(T)p<₀.₀₅, MIA-1, A549, and T47D with RSV-LacZ vs MIA-1, A549,and T47D with REP-LacZ, chi square.

CAPAN-1 cells did not turn blue even with the RSV-LacZ construct whileall of the other cells transfected with RSV-LacZ developed blue colorafter X-gal staining ensuring that they were adequately transfected.

Detection of β-galactosidase gene expression using a luminometer:PANC-1, CAPAN-1, MIA-1, and A549 cells, which were transfected witheither RIP-LacZ or RSV-LacZ, were also subjected to beta-galactosidasereporter gene assay (Tropix, Bedford, Mass.). The assay was carried outin triplicate. Protein levels were determined using Bradford's proteinassay (Sigrna, St. Louis, Mo.). Results were represented in light unitsand adjusted for protein content.

As shown in Table 2, transfection of RIP-LacZ resulted in a significantincrease in beta-galactosidase protein levels in both PANC-1 and CAPAN-1cells compared to A549 and MIA-1 cells. TABLE 2 Construct PANC-1 CAPAN-1A549 MIA-1 RSV-LacZ 9.7 × 10⁵* 9.1 × 10⁵* 9.2 × 10⁵ 9.5 × 10⁵ HV 1.3 ×10⁵* 1.0 × 10⁵* 1.2 × 10⁵ 1.1 × 10⁵ Untransfected 1.2 × 10⁵* 1.1 × 10⁵*1.3 × 10⁵ 1.3 × 10⁵ RIP-LacZ 3.8 × 10^(5Δ) 4.2 × 10^(5Δ) 1.2 × 10⁵ 1.2 ×10⁵

Table 2: Beta-galactosidase activity in PANC-1, CAPAN-1, A549, and MIA-1cells thirty-six hours post-transfection with either RSV-LacZ, a hollowvector (HV), nothing or RIP-LacZ. Data represented in light units andadjusted for protein content. n=6, ^(Δ)p<0.05, ANOVA, *p=NS.

All cell types demonstrated and equal amount of beta-galactosidase aftertransfection with RSV-LacZ ensuring that both A549 and MIA-1 cells wereadequately transfected. Background light units were also equal betweencell types (Table 2).

Ganciclovir dose response curve for cells in culture: PANC-1, CAPAN-1MIA-1 and A549 cells were plated into a ninety-six well plate at adensity of 5,000 cells per well and given between 0 to 500 μg/ml of GCVto determine a dose response curve to GCV alone. The cells were treatedfor five days and cell viability was ascertained with an MTS assay.Results were plotted on a graph as OD 490 nm vs. GCV concentration.

PANC-1, CAPAN-1, A549, and MIA-1 cells were transfected with a controlvector, RSV-LacZ, or RIP-LacZ plasmid for 36 hours. Cells were washedand collected. The β-galactosidase activity from each transfection wasdetermined using a luminometer. Data was obtained in light units, andwas corrected for protein content (FIG. 5A). The vertical axis is inlight units per μg/ml protein. As shown in FIG. 5A, GCV dosages greaterthan 50 μg/ml significantly decreased untransfected PANC-1 and A549 cellOD 490 compared to cells that received no GCV (n=8, p<0.05)Specifically, FIG. 5A is a dose response curve for PANC-1 cells givenGCV. Note that at concentrations of GCV greater than 50 μg/ml, asignificant decrease in cell survival was observed, n=8, *p<0.05,unpaired Student t-test. This determined that 20 μg/ml is the maximumdose of GCV one can use to treat PANC-1 cells in culture, and was usedfor subsequent experiments. PANC-1, CAPAN-1, A549, and MIA-1 cells weretransfected with RIP-tk, U-tk (tk with a ubiquitous promoter), HV(hollow vector), or UT (untransformed) for 36 hours in order to assaycell death. All cells were treated for five days with either 15 or 20:g/ml of GCV. Cell viability was determined by an MTS assay (Promega,Madison, Wis.). The data is shown in FIG. 5B. The vertical axis ispercent cell death. As shown in FIG. 5B, GCV dosages greater than 20μg/ml significantly decreased untransfected CAPAN-1 and MIA-1 cell OD490 compared to cells that received no GCV (n=16, p<0.05). Specifically,FIG. 5B is a dose response curve for CAPAN-1 cells given GCV. Note thatat concentrations of GCV greater than 20 μg/ml, a significant decreasein cell survival was observed, n=8, *p<0.05, unpaired Student t-test.This determined that 15 μg/ml is the maximum dose of GCV one can use totreat CAPAN-1 cells in culture, and was used for subsequent experiments.

Treatment of transfected cells with ganciclovir: PANC-1, CAPAN-1, MIA-1and A549 cells were transfected with either RIP-tk gene, a hollow vectorcontrol (HV), or with thymidine kinase construct that was driven by aubiquitous promoter (U-tk) (provided by Dr. Fanco DeMayo, Departments ofCell Biology and Pediatrics, Baylor College of Medicine). Twenty-fourhours post transfection the cells were re-plated into ninety-six wellplates at a density of 5,000 cells per well, and subjected to GCVtreatment at a dose of 15-20 Ag/ml. The media was refreshed daily. Cellviability was determined by using an MTS assay (Promega, Madison, Wis.)read at an absorbency of 490 nm. Percent cell death was calculatedutilizing the following formula: $\frac{A - B}{A} \times 100$where A is the absorbency at 490 nm of transfected cells not treatedwith GCV and B is the absorbency at 490 nm of transfected cells treatedwith GCV. Cell survival was calculated by subtracting the percent celldeath from one hundred. Untransfected cells (UT) were also treated withganciclovir to determine the effect of GCV alone on cell death.

As shown in Table 3, in cells transfected with the RIP-tk geneticconstruct both the PANC-1 and CAPAN-1 cells demonstrated a significantincrease in cell death; 31±0.1% and 13±0.1% (Table 4). Both MIA-1 andA549 cells failed to show any significant increase in cell death withthe RIP-tk gene. TABLE 3 RIP-tk U-tk HV UT PANC-1 31 ± 0.1^(Δ)* 25 ± 0.30 ± 0.1 4 ± 0.1 A549  2 ± 0.1 30 ± 0.1 2 ± 0.1 4 ± 0.1 CAPAN-1 13 ±0.1^(T7)  8 ± 0.1 0 ± 0.3 4 ± 0.1 MIA-1  0 ± 0.1  7 ± 0.1 0 ± 0.1 0 ±0.1

Table 3: Percent cell death for PANC-1, CAPAN-1, A549, and MIA-1 cellstransfected with RIP-tk, U-tk (tk with a ubiquitous promoter), HV(hollow vector) and UT (untransfected). All cells were treated for fivedays with 20: g/ml of GCV. Cell viability ascertained by an MTS assay(Promega, Madison, Wis.). Ap<0.05, PANC-1 with RIP-tk vs. A549 and MIA-1with RIP-tk and PANC-1 with HV and UT. *p=NS, PANC-1 with RIP-tk vs.PANC-1 with U-tk and A549 with U-tk. ^(T)p<₀.₀₅, CAPAN-1 with RIP-tk vs.A5491 and MIA-1 with RIP-tk and CAPAN-1 with HV and UT. ⁷p=NS, CAPAN-1with RIP-tk vs. CAPAN-1 with U-tk and MIA-1 with U-tk.

None of the cell types demonstrated any significant increase in celldeath following transfection with the negative controls, hollow vector(HV) or with GCV treatment alone (UT) (Table 3). All cell lines with thepositive control U-tk demonstrated a significant increase in cell death(Table 3). The data suggest that all cell types were sufficientlytransfected and susceptible to the cytotoxic effects of tk followed byGCV.

RNA isolation and RT-PCR Analysis of RIP transcription factors PDX-1 andBETA-2: PANC-1, CAPAN-1, and MIA-1 total RNA was extracted using RNAzo1™(Tel-Test, INC., Friendswood, Tex.). Cells were grown to confluence inlarge vented flasks. Media was removed and 5 ml of RNAzolTM was added.Cells were removed and placed on ice for fifteen minutes. 500:1 ofchloroform was added and the cells were spun for fifteen minutes at10,000 rpm at 4° C. The aqueous phase was removed and mixed with anequal volume of isopropanol and placed at 4° C. for fifteen minutes andthen spun for fifteen minutes at 15,000 rpm at 4° C. The pellet was thenwashed with 70% ethanol and re-spun at 15,000 rpm, and then dried.

Reverse transcriptase polymerase chain reaction (RT-PCR) was performedusing SUPERSCRIPT™ Preamplification System for First Strand cDNASynthesis kit (Gibco-BRL, Bethesda Md.). Primers specific for humanPDX-1 RNA (forward bps 192-210, gggaacgccacacagtgcca (SEQ ID NO: 10) andreverse bps 644-624, gtaccctttccgtcgacctg (SEQ ID NO: 11) and primersspecific for both human and mouse BETA-2 RNA (forward bps 944-964,cgccgagtttgaaaaaaatt; SEQ ID NO: 7, and reverse bps 1227-1207,tttttccgacggaagacatt; SEQ ID NO: 8), (BETA-2 primers were provided byDr. Ming-Jer Tsai, Department of Cell Biology, Baylor College ofMedicine) were used. Standard β-actin primers were used as controls. PCRprogram for all three primers was as following: 2 min 94° C.; thenthirty cycles of 1 min 94° C., I min 55° C., and 1 min 72° C.; and 5 min72° C. for finishing. 5 ll of the PCR reaction was run on gelelectrophoresis.

A 400 bp band identified a positive for PDX-1 and a 300 bp bandidentified a positive for BETA-2. NIT-1 cell (mouse 3-cell adenoma cellline) RNA was used as a positive control for the BETA-2 25 primers.

PDX-1 RNA was identified in PANC-1 and CAPAN-1 cells (FIG. 5C). BETA-2RNA was not found. BETA-2 RNA was identified in a NIT-1 cell linecontrol (FIG. 5C). MIA-1 cells contained no RNA for either PDX-1 orBETA-2 (FIG. 5C). Specifically, FIG. 5C is a gel electrophoresis forRT-PCR products of PANC-1, CAPAN-1 and MIA-1 RNA with primers specificfor PDX-1 (odd lanes) and BETA-2 (even lanes).

Nuclear extracts and electrophoretic mobility-shift assays (EMSA):Nuclear extracts were isolated as described by Olson et al. (Olson L, etal., 12(2) MOL. ENDOCRINOL. 207-219 (1998)). Protein levels weredetermined using Bradford's protein assay (Sigma, St. Louis, Mo.). Adouble-stranded oligodeoxynucleotide probe to the PDX-1 site within RIP(bp 350-381 on RIP TTGGCCATCGTCTGATCCAACCCTTAATGGGAC; SEQ ID NO: 9) waslabeled with α³²P dGTP by filling overhanging 5′ ends with Superscript™(Gibco-BRL, Bethesda MD.) Binding reactions were performed with 1.5: gof protein and 1:1 hot probe per lane on a 5% acrylamide gel with eithernuclear extract alone, 100X cold wild type probe, or 100X cold mutatedprobe to assess protein binding (the mutated probe contained the samesequence of RIP as the wild type probe except the PDX-1 binding site wasaltered from CTTAAT (SEQ ID NO: 4) to CTCCCC (SEQ ID NO: 12)).Supershift analysis for PDX-1 binding activity was performed by theaddition of 1:1 of anti-N-terminal XIHbox8 antibody (provided by Dr.Christopher Wright, Department of Medicine, Vanderbilt University Schoolof Medicine).

Nuclear extract from PANC-1 and CAPAN-1 cells bound to the RIP primercontaining a PDX-1 binding site. The binding was effectively inhibitedby cold primer and not by cold mutated primer (same sequence of RIP withthe PDX-1 binding site altered, CTTAAT (SEQ ID NO: 4) to ATATAC (SEQ IDNO: 13)). A supershift was observed using the XIHbox8 antibody (FIGS. 6Aand 6B).

Results are shown in FIGS. 6A and 6B. Specifically, FIG. 6A is an EMSAof PANC-1 nuclear extract mixed with ∀³²P dGTP labeled RIP primercontaining a PDX-1 binding site (CTTAAT; SEQ ID NO: 4). Lane 1 isnuclear extract with hot probe alone. Arrow denotes prominent band. Lane2 is nuclear extract with hot probe and I OOX competitor cold probe.Note that a band is no longer present. Lane 3 is nuclear extract withhot probe and 10 OX competitor cold mutated probe (PDX-1 site in RIP ismutated to ATATAC (SEQ ID NO: 13). Note that a band is present. Themutated probe is unable to compete the hot probe off the nuclearextract. Lane 4 is nuclear extract with hot probe plus 1:1 of XIHbox8antibody (specific for PDX-1). The arrow denotes the supershift.

FIG. 6B is an EMSA of CAPAN-1 nuclear extract mixed with ∀³²P dGTPlabeled RIP primer containing a PDX-1 binding site (CTTAAT; SEQ ID NO:4). Lane 1 is probe alone. Lane 2 is nuclear extract with hot probealone. Arrow denotes prominent band. Lane 3 is nuclear extract with hotprobe and lOOX competitor cold probe. Note that a band is no longerpresent. Lane 4 is nuclear extract with hot probe plus 1:1 of XIHbox8antibody (specific for PDX-1). The arrow denotes the supershift. Lane 5is nuclear extract with hot probe and lOOX competitor cold mutated probe(PDX-1 site in RIP is mutated to ATATAC; SEQ ID NO: 13). Note that aband is present. The mutated probe is unable to compete the hot probeoff the nuclear extract.

Generation of mutated RIP-LacZ: Two oligonucleotides were designed toPCR amplify RIP and mutate the PDX-1 binding site found at 430. Thesequence was mutated from CTTAAT to ATATAC The 5′ oligonucleotidecontained a HindlIl binding site (sequence GAAAGCTTTCTGCTTTCCTTCTACCTC(SEQ ID NO: 14) and the 3′ oligonucleotide contained a BglII restrictionsite (in bold, SEQ ID NO: 15, sequenceTCTAGAGCTTGGACTTTGCTGTTTGTCCCGTATATGGTGGATCAGCAG). The restriction siteswere added to facilitate construct formation. The PCR program was asfollowing: 2 min 94° C.; then thirty cycles of 1 min 94° C., 1 min 55°C., and 1 min 72° C.; and 5 min 72° C. for finishing. 5:1 of the PCRreaction was run on gel electrophoresis. A sole 450 bp band identifiedthe PCR product which was then ligated into a TA cloning vector(Invitrogen, Carlsbad, Calif.). The ligation was confirmed with EcoRIand the identification of a 450 bp band. Gene sequencing of the vectorwas performed (Core Sequencing Facility, Baylor College of Medicine).

To create the mutated RIP-LacZ construct the mutated RIP was isolatedfrom the TA cloning vector by digesting with BglII and Hindll. Theisolated mutated RIP promoter was then ligated with the PD46.21 vectorafter it was digested with Hindffi and BamHI. This ligation was verifiedwith HindlIl and Cla I with a 1.5 Kb band.

Comparison of mutated RIP-LacZ and wild type RIP-LacZ: PANC-1 andCAPAN-1 cells were transfected with either the mutated RIP-LacZ or thewild type RIP-LacZ as described above. Thirty-six hours post transfectedthe cells were subjected to beta-galactosidase reporter gene assay(Tropix, Bedford, Mass.). The assay was carried out in triplicate.Protein levels were determined using Bradford's protein assay (Sigma,St. Louis, Mo.). Results were represented in light units and adjustedfor protein content.

As shown in Table 4, transfection of wild type RIP-LacZ resulted in asignificant increase in beta-galactosidase protein levels in both PANC-1and CAPAN-1 cells as compared to the mutated RIP-LacZ. TABLE 4 ConstructPANC-1 CAPAN-1 RIP-LacZ 1.0 × 10⁵* 1.4 × 10^(5Δ) mRIP-LacZ 2.5 × 10⁴ 4.8× 10⁴

Table 4: Beta-galactosidase activity in PANC-1 and CAPAN-1 cellsthirty-six hours post-transfection with either RIP-LacZ or a mutatedRIP-LacZ (mRIP-LacZ). Data represented in light units and adjusted forprotein content (n=8) *p<0.05, PANC-1 RIP-LacZ vs PANC-1 mRIP-LacZ, chisquare. ^(Δ)p<0.05, CAPAN-1 RIP-LacZ vs CAPAN-1 mRIP-LacZ, unpairedStudent's t-test.

Treatment of human ductal pancreatic adenocarcinoma tumors in vivo:Female ICR/scid mice, age 6-10 weeks old underwent intraperitoneal (IP)injections of 5×10⁵ PANC-1 cells or the negative control PBS asdescribed (Schwartz R E, et al., 126(3) SURGERY 562-567 (1999)). At day21 the mice were randomized to receive the RIP-tk gene (n=9), theRIP-LacZ gene (n=9), or PBS (n=6), followed by 7 days of GCV (40 mg/kgIP). Six mice received seven days of PBS IP. Genes complexed with 20 mMextruded DOTAP:cholesterol were delivered IP for in vivo gene delivery(Smyth-Templeton N, et al., 15 NATURE BIOTECHNOL. 647-652 25 (1997)).

Three additional mice inoculated with PANC-1 cells received the RIP-LacZgene and were sacrificed thirty-six hours later. Brain, heart, lung,liver, small bowel, spleen, kidney, and pancreas were fixed in 5%glutaraldehyde for one hour, stained with X-gal staining solution fortwenty-four hours, and then counterstained with nuclear fast red.Analysis of treatment and gene placement consisted of size of the tumorand presence of blue color after X-gal staining.

Twenty-one days post tumor the mice were randomized (n=6 per group) asfollows: 1) PANC-1, RIP-tk and GCV; 2) PANC-1, RIP-LacZ and GCV; 3)PANC-1 and GCV only; and 4) PANC-1 and PBS only. GCV was given at 40mg/kg IP BID for 7 days to activate the tk. Additional injected micewith tumors (n=3) received RIP-LacZ and their tissues were fixed andstained with X-gal. Study endpoints consisted of monitoring tumor sizeand the presence of blue cells after X-gal staining.

Results are shown in FIG. 8. FIG. 8 is a photograph of X-gal stainedPANC-1 cells. PANC-1 cells were effectively targeted with the RIP-LacZgene in vivo; only the PANC-1 cells stained blue. The in vivo deliveryof RIP-tk in combination with GCV resulted in a significant decrease intumor burden in mice; the combination killed all PANC-1 tumors in eightof nine mice (p<0.05 compared to all other groups, ANOVA). Mice thatreceived PANC-1 cells and treated with RIP-LacZ (the vector control) andmice treated with only GCV developed large peri-pancreaticintraperitoneal tumors.

Statistics: All cells used in the experiment were compared using bothANOVA and chi square after X-gal staining. The light units were adjustedfor protein content and compared using an unpaired Student's t-test.Cell survival was compared between different constructs and cell lineswith ANOVA. Tumor sizes were compared using ANOVA. P<0.05 representedsignificance.

EXAMPLE 3 Human Dectal Pancreatic Adenocarcinoma Cells that ExpressPDX-1 can be Targeted with a RIP-tk Gene

RIP-LacZ was created and transfected into CAPAN-1 (C-1) and MIA-1 (M-1)cell lines. RIP-tk was created and transfected into C-1 and M-1 celllines. Tk driven by a ubiquitous promoter (U-tk), a hollow vector (HV)and untransfected cells (UT) were used as controls. Cells were treatedfor five days with 15 μg/ml of ganciclovir and cell viability wasassessed during a MTS assay. RT-PCR was performed on C-1 and M-1 RNAwith primers specific for PDX-1 and BETA-2. Nuclear extract from C-1cells was subjected to a gel shift assay with an antibody to PDX-1. APDX-1 binding site on RIP was mutated (mRIP) with PCR and a mRIP-LacZconstruct was created and transfected in C-1 cells. Results are shown inTable 5. TABLE 5 Results: LacZ expression (LU) Percent Cell Death RIPZRSVZ mRWZ RIP-tk U-tk HV UT C-1 4.2 × 10⁵ 9.1 × 10⁵ 1.4 × 10^(5T) 13 ±.1* 8 ± .1 0 ± .1 4 ± .1 M-1 1.2 × 10⁵ 9.5 × 10⁵  0 ± .1 7 ± .1 0 ± .1 0± .1

Table 5: ^(Δ)p<0.05 C-1 RIP-LacZ vs. M-1 RIP-LacZ, and ^(T)p<0.05 C-1mRIP-LacZ vs. C-1 RIP-LacZ, students t-test. *p<0.05 C-1 RIP-tk vs. M-1RIP-tk and negative controls, ANOVA.

Neither cell type contained RNA for BETA-2, however C-1 cells containedRNA for PDX-1. The nuclear extract of C-1 cells bound to the PDX-1sequence of RIP (CTTAAT) and a super shift was observed with the PDX-1antibody.

The data confirm that RIP can drive the expression of a gene in humanPDA cells. Additionally, the transcription factor PDX-1 is usefuil forpromoter activation in human PDA cells.

EXAMPLE 4 Selective Human Pancreatic Cnacer Targeting Using an RIP-tkConstruct (in vitro Data).

This example further demonstrates that a human pancreatic ductalcarcinoma cell line (PANC-1) may be selectively targeted using the ratinsulin promoter (RIP) and that PANC-1 cytotoxicity may be induced usingRIP with the thymidine kinase gene (tk).

0.502 kb of RIP was ligated to LacZ and transfected into human cancercell lines: PANC-1, lung (A549), and breast (T47D), in vitro. Analysisof LacZ gene expression was by X-gal staining. RIP was also ligated totk (REP-tk) and transfected into PANC-1 and A549 cells.

Untransfected cells (UT) and a hollow vector (HV) served as negativecontrols, while tk under the control of a ubiquitous promoter (M-tk)served as a positive control. Cells were treated daily with ganciclovir(GCV). Viability was measured on day six using an MTS assay. Results areshown in Table 6. TABLE 6 Percent Cell Death Cell LacZ % RIP-tk M-tk HVUT PANC-1 30 31 ± .1⁶⁶ 25 ± 3 0 ± .1 4 ± .1 A549  0  2 ± .1 30 ± .1 2 ±.1 4 ± .1 T47D <1

Table 6:* p<0.05, ^(Δ)p<0.0001 compared to PANC-1 (HV & UT) and A549(RIP-tk, HV, & UT), p=NS compared to M-tk for both cell lines, ANOVA(n=32).

The resulting data confirm that RIP can drive the expression of a genein human pancreatic ductal carcinoma (PANC-1) cells. The RIP-tk generesulted in PANC-1 specific cytotoxicity as effective as M-tk. Thus, itcan be concluded that PANC-1 can be targeted with RIP.

EXAMPLE 5 β-Cell Specific Cytotoxicity using RIP-tk Construct (in vitroData)

This example demonstrates the use of the rat insulin promoter (RIP) willresult in the β-cell specific expression of a transfected gene and thatone can induce β-cell specific cytotoxicity using RIP with the thymidinekinase gene (tk).

0.502 kb of RIP was ligated to the reporter gene LacZ and transfectedinto several cell lines: insulinoma (NIT-1), embryonic carcinoma (F9),fibroblast (3T3), and lung (H441) cells in vitro. X-gal staininganalyzed the LacZ gene where blue nuclei represented cellularexpression. RIP was ligated to tk and transfected into NIT-1 and F9cells. A hollow vector (V) and tk under the control of a ubiquitouspromoter (MC-1-tk) were used as negative and positive controls,respectively. The cells were treated daily with ganciclovir (GCV). Cellviability was ascertained on day six using an MTS assay. Results areshown in Table 7. TABLE 7 Results: NIT-1 F9 3T3 H441 N = 16 per celltype LacZ Expression ++++ − − −

The bar graphs in FIG. 9 depict the cell survival percentages of NIT-1and F9 cell lines following transfection and GCV treatment. The cellswere transected with vectors encoding either RIP-tk (test), tk under thecontrol of a ubiquitous promoter as a positive control (MC-1-tk), and tkin a hollow vector as a negative control (V), followed by treatment withGCV in the indicated concentration. (*p <0.05 via student t-test, n=48per construct.)

The data indicates that the RIP is a β-cell specific promoter. A β-cellspecific and GCV dose dependent decrease in NIT-1 cell survival wasdemonstrated with the RIP-tk gene. F9 cell death was shown using theMC-1-tk gene at similar doses of GCV, but not with the RIP-tk geneindicating that β-cell targeted cell death can be accomplished by thetissue specific expression of tk by RIP.

EXAMPLE 6 NIT Specific Killing with RIP-tk (in vitro Data)

This is another example of NIT targeted cytotoxicity in vitro. Twogenetic constructs were created, RIP-LacZ and RIP-tk. The RIP-LacZconstruct was delivered into NIT-1 cells and control cell lines. OnlyNIT-1 cells stained blue following X-gal staining, demonstrating NIT-1specificity of RIP. Secondly, NIT-1 cells and control cells weretransfected with RIP-tk construct, then treated with GCV. A highlysignificant ablation of only NIT-1 cells was achieved, demonstrating theNIT-1 specific killing effect of RIP-tk (Tirone et al., ANNALS SURGERY,in press).

Using RNA isolated from NIT-1 cells, the presence of the RIPtranscription factor, BETA-2, was demonstrated using RT-PCR. Thisobservation supports the finding that the transcription factor BETA-2regulates the effect of RIP-tk in an insulinoma cell line.

EXAMPLE 7 Enhancement of the Cytotoxic Effect of RIP-tk by RIPTranscriptional Factors BETA-2, GATA4, AND E47

BETA-2 forms heterodimers with E47 and GATA4 and enhances RIP drivengene expression in (β-cells (German M, et al., 75J MOL MED 327-340(1997)). The cytotoxic effect of RIP transcription factors BETA-2,PDX-1, GATA4, and E47 in NIT-1 cells is assessed by co-transfectingthese transcription factors with either RIP-tk or RIP-luciferase geneticconstructs. Both cytotoxicity and luciferase activity is quantified.

EXAMPLE 8 Alterations in Insulin Secretion in the SSTR-5 Knock out Mouseusing Isolated Perfused MousePancreas Model

SSTR-5 knock out (KO) mice 3-months-old (n=6), SSTR-5 (somatostatinsubtype receptor 5) KO mice 12-months-old (n=8), and age matched wildtype (wt) controls (n=6 and n=8, respectively) were screened by Southernblots to determine their genotype. KO and wt mice 3 and 12 months old(n=4 per group) underwent histological examination of islets by anindependent pathologist. Pancreata were isolated as described elsewherewith the exception that all pancreata remained in situ throughout theirperfusion. (Lenten S, 235(4) AM. J. PHYSIOL. E391-E3400 (1979)). Singlepass perfusion of isolated pancreata was performed using a Krebs bufferequilibrated with 95% O₂/ 5% CO₂ containing 70 mg % glucose for fiveminutes (basal) and 300 mg % glucose for an addition twenty-six minutes(stimulated). Basal insulin secretion, and glucose stimulated first andsecond phase insulin secretion were compared by calculating the areaunder the secretion vs. time curve utilizing the trapezoidal rule.(Chiou WL, 6(6) J. PHARMACOKINET. & BIOPHARM. 539-546 (1978)). Insulinwas measured in duplicate using, ELISA presented as mean ±SEM in pg/ml.Statistical analysis was by ANOVA.

Histological sections of islets suggest there is no difference in isletcell morphology between 3-month-old KO and 3-month-old wt mice or12-month-old KO and 12-month-old wt mice. There were no differences inweight between KO and aged match controls. There were no differences inbasal insulin secretion between any of the mice. Additionally, glucosestimulation caused in a significant increase in insulin secretioncompared to basal in all mice. Three-month-old KO mice demonstrated ablunted first phase that was significant compared to all other mice(Table 8 and FIG. 7). Twelve-month-old KO mice demonstrated asignificant augmentation of both first phase and second phase comparedto all other groups Results are shown in Table 8 and FIG. 7. TABLE 8 KO3 mo WT 3 mo KO 12 mo WT 12 mo Basal 254 ± 9 279 ± 5 301 ± 11 266 ± 101st Phase 248 ± 10*♦ 318 ± 7^(♦) 590 ± 17^(♦) 417 ± 19^(♦) 2nd Phase 435± 9^(♦) 457 ± 9^(♦) 767 ± 18^(+♦) 417 ± 9^(♦)

Table 8: Average insulin levels in pg/ml. Basal insulin levels acrossall groups were similar (p=NS). ♦All mice experienced significantglucose stimulated insulin secretion compared to basal levels, p<0.05.*Three-month-old KO mice demonstrated a blunted first phase compared toall other groups, p<0.05. Twelve month-old KO mice demonstrated anincreased first phase compared to all other groups, p<0.05. +Twelvemonth KO mice demonstrated an increased second phase compared to allother groups, p<0.05.

FIG. 7 is a glucose stimulated insulin versus time curve for 3-month-oldand 12-month-old SSTR-5 KO and wt mice. Glucose stimulation with 300 mg% glucose started at time zero. There was no significant difference inbasal insulin levels across all groups and all animals had a significantincrease in insulin secretion compared to basal levels. Note the bluntedfirst phase response seen in 3-month-old KO mice and the augmented firstand second phase response seen in 12-month-old KO mice.

EXAMPLE 9: Intraislet Somatostatin

Preliminary data suggest the presence of delta-to-beta cell endocrineaxis within the islet in which intraislet somatostatin inhibits insulinsecretion. The purpose of this example is to prove the followinghypotheses: 1) intraislet somatostatin inhibits insulin secretion via adelta-to-beta cell endocrine axis in the human, rat and mouse pancreasand that the effect is glucose-dependent; 2) the somatostatin receptorsubtype responsible for the inhibition of insulin is species-specific;and 3) genetic ablation of the somatostatin receptor subtype-5 willalter insulin secretion and glucose homeostasis in the mouse.

The effect of intraislet somatostatin is determined by examining theinsulin response to immunoneutralization of intraislet somatostatin withantibodies and FAb fragments of antibodies directed against somatostatinin isolated perfused human, rat and mouse pancreas models. Electronmicroscopy is used to help to determine the compartment ofimmunoneutralization.

The somatostatin receptor subtype responsible for the inhibition ofinsulin is determined by examining the response of insulin secretion toinfusions of specific somatostatin receptor subtype agonists in thesemodels. Immunohistochemistry is performed using polyclonal antibodiesdirected against SSTR 1-5 to determine which receptor subtypes arepresent in the human, rat and mouse pancreas. The somatostatin receptorsubtype-5 appears responsible for the inhibition of mouse insulinsecretion. Thus, two models are developed using state-of-the-arttransgenic techniques: the first is a total somatostatin receptorsubtype-5 gene ablation model and the second is a β-cell-specificsomatostatin receptor subtype-5 gene ablation model. In vivo and invitro physiology studies are performed in these mice to determine theeffect of genetically altering the delta-to-beta cell endocrine axis oninsulin secretion and glucose homeostasis. The pancreas of thegene-ablated mice are studied using immunohistochemistry with antibodiesdirected against the somatostatin receptor subtypes to determine if thesomatostatin receptor subtypes are altered in the islets of the geneablated mice.

Other studies involve a β-cell specific BETA-2 knockout mouse model andpromoter analysis of the SSTR-5 gene including the role of thetranscription factor BETA-2 in activation of the SSTR-5 gene and insulinexpression. The results will elucidate physiologic mechanisms regulatinginsulin secretion and determine whether there are species differences inthis regulation. Furthermore, pathophysiologic consequences togenetically altering these mechanisms may be determined.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including the priority documents and all U.S. and foreign patents andpatent applications, are specifically and entirely incorporated byreference. It is intended that the specification and examples beconsidered exemplary only, with the true scope and spirit of theinvention indicated by the following claims.

1-139. (canceled)
 140. A method of treating hyperinsulinemia in asubject, comprising: a) directly administering to a subject a nucleicacid comprising SEQ ID NO: 1operatively coupled to a cytotoxic gene,wherein the cytotoxic gene is thereby expressed in a pancreatic betacell, b) administering a pro-drug to said subject, wherein the prodrugis converted to a cytotoxic compound by the action of the proteinencoded by said cytotoxic gene, thereby killing the beta cell andtreating hyperinsulinemia.
 141. The method of claim 1, where thecytotoxic gene is the thymidine kinase gene.
 142. The method of claim 1,where the cytotoxic gene is the thymidine kinase gene and the prodrug isacyclovir, ganciclovir, FIAU or 6-methoxypurine arabinoside.
 143. Amethod of killing a beta cell that over-expresses insulin in a subject,the method comprising: a) administering to a subject a nucleic acidcomprising SEQ ID NO: 1 operatively coupled to a cytotoxic gene, whereinthe cytotoxic gene is thereby expressed in a beta cell thatover-expresses insulin, b) administering a pro-drug to said subject,wherein the prodrug is converted to a cytotoxic compound by the actionof the protein encoded by said cytotoxic gene and thereby killing thebeta cell that over-expresses insulin.
 144. The method of claim 143,where the cytotoxic gene is the thymidine kinase gene.
 145. The methodof claim 143, where the cytotoxic gene is the thymidine kinase gene andthe prodrug is acyclovir, ganciclovir, FIAU or 6-methoxypurinearabinoside.
 146. The method of claim 145, wherein the administration issystemic.
 147. The method of claim 145, wherein the administration is bydirect administration at the site of the beta cell.
 148. A method oftargeting a beta cell in a subject, the method comprising administeringto a subject an expression vector comprising SEQ ID NO: 1 operativelycoupled to a gene, wherein the gene is thereby expressed in a pancreaticbeta cell in said subject.
 149. The method of claim 148, wherein gene isa cytotoxic gene.
 150. A nucleic acid comprising a sequence that has atleast 95% identity to SEQ ID NO: 1 operatively coupled to a cytotoxicgene.
 151. The nucleic acid of claim 150, comprising a sequence with atleast 98% identity to SEQ ID NO:
 1. 152. The nucleic acid of claim 150,comprising SEQ ID NO: 1.